Method for increasing plant yield, and yield improving compositions

ABSTRACT

The present invention relates to a method of increasing yield of a plant. This method involves applying to a plant and/or area of cultivation thiophanate methyl and an isolated hypersensitive response elicitor protein or polypeptide fragment, wherein said applying is carried out under conditions effective to induce a synergistic yield from the plant. The present invention also relates to a composition comprising a liquid or solid carrier, thiophanate methyl, and an isolated hypersensitive response elicitor protein or polypeptide fragment.

This application claims the priority benefit of U.S. Provisional PatentApplication Ser. No. 61/581,819, filed Dec. 30, 2011, which is herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to a method for increasing yield of a plant, andcompositions for improving yield of a plant.

BACKGROUND OF THE INVENTION

In crop production, there is a continuous need for agriculturalcompositions and treatments that improve the health of plants. Healthierplants are desirable since they result in better yields and/or a betterquality of the plants or crops. Healthier plants also better resistbiotic and/or abiotic stress. A high resistance against biotic stressesin turn allows a lower quantity of applied pesticides, which can thenslow down the development of resistances against the respectivepesticides.

Hypersensitive response (“HR”) elicitor proteins (also known as harpinproteins) elicit disease resistance in plants and increase plant growth.U.S. Pat. No. 6,277,814 describes a method of enhancing growth inplants, which involves applying an HR elicitor polypeptide or protein ina non-infectious form to plants or plant seeds under conditions toimpart enhanced growth to the plants or to plants grown from the plantseeds. These methods are carried out to affect any form of plant growthenhancement or promotion, including greater yield, increased quantity ofseeds produced, increased percentage of seeds germinated, increasedplant size, greater biomass, more and bigger fruit, earlier fruitcoloration, and earlier fruit and plant maturation. Early germinationand early maturation permit crops to be grown in areas where shortgrowing seasons would otherwise preclude their growth in that locale.Increased percentage of seed germination results in improved crop standsand more efficient seed use. Greater yield, increased size, and enhancedbiomass production allow greater revenue generation from a given plot ofland.

Agricultural compositions containing isolated HR elicitor proteins havebeen used to improve plant health and increase yield. For example, PCTPublication No. WO 04/057957 to Wei teaches increasing the efficacy ofan agricultural chemical by applying at least one agricultural chemicaland at least one HR elicitor protein or polypeptide to a plant or plantseed under conditions effective to increase the efficacy of theagricultural chemical.

There is a need for agricultural compositions and plant treatments thatoffer a synergistic effect on plant yield, meaning the combined effectsof the agents is greater than the additive effects of each individualagent.

The present invention is directed to overcoming these limitations in theart.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to a method of increasingyield of a plant. This method involves applying to a plant and/or areaof cultivation thiophanate methyl and an isolated hypersensitiveresponse elicitor protein or polypeptide fragment, wherein said applyingis carried out under conditions effective to induce a synergistic yieldincrease from the plant.

Another aspect of the present invention relates to a compositioncomprising a liquid or solid carrier, thiophanate methyl, and anisolated hypersensitive response elicitor protein or polypeptidefragment.

The present invention relates to increasing the yield of a plant byapplication of agents that when applied together or in sequence have asynergistic effect on plant yield. The agents include at least thefungicide thiophanate methyl (or another benzimidazole fungicide) and anisolated hypersensitive response elicitor protein or polypeptidefragment.

As demonstrated in the accompanying Examples, the combined effects ofthiophanate methyl and an isolated hypersensitive response elicitorpolypeptide designated PHC-351 (Plant Health Care, Inc., Pittsburgh,Pa.) demonstrate in pinto beans and peanut a surprisingly synergisticeffect of the two agents on plant yields.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a table showing the results from an experiment testing thecontrol of white mold in pinto bean plants by applying to the plantsPHC-351 and thiophanate methyl treatments individually and incombination. The results show the calculation of percent increase inyield in pinto bean plants.

FIG. 2 is a table showing the results of an experiment testing thecontrol of white mold in peanut plants by applying to the plants PHC-351and thiophanate methyl treatments individually and in combination. Theresults show the calculation of percent increase in yield.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of increasing yield in aplant, and compositions useful in increasing plant yield.

According to the method of the present invention, the fungicidethiophanate methyl and an isolated hypersensitive response elicitorprotein or polypeptide fragment are applied to a plant and/or area ofcultivation under conditions effective to induce a synergistic yieldfrom the plant in response to said applying.

The phrase “area of cultivation” means any type of environment, soil,area, or material where the plant is growing or intended to grow.

As used herein, the term “plant” includes all parts of a plant,including herbaceous vegetation, leaves, roots, stems, floralstructures, pollen, etc. In addition, “plant” means all plants and,particularly, plants of economic importance. Plants may be categorizedinto agricultural, silvicultural, ornamental, and horticultural plants,based on their human use and/or consumption. In addition, “plants”include natural or wildtype plants, and plants that have beengenetically modified.

“Agricultural” plants are plants of which a part or all is harvested orcultivated on a commercial scale or which serve as an important sourceof feed, food, fibers (e.g., cotton and linen), combustibles (e.g.,wood, bioethanol, biodiesel, and biomass) or other chemical compounds.Agricultural plants also include vegetables. Thus, agricultural plantsinclude cereals (e.g., wheat, rye, barley, triticale, oats, sorghum, andrice); beet (e.g., sugar beet or fodder beet); leguminous plants (e.g.,beans, lentils, peas, alfalfa, and soybean); oil plants (e.g., rape,oil-seed rape, canola, juncea (Brassica juncea), linseed, mustard,olive, sunflower, cocoa bean, castor oil plants, oil palms, ground nuts,and soybean); cucurbits (e.g., squash, cucumber, and melon); fiberplants (e.g., cotton, flax, hemp, and jute); vegetables (e.g.,cucumbers, spinach, lettuce, asparagus, cabbages, carrots, radish,turnip, celery, chicory, endive, brussel sprouts, parsnip, cauliflower,broccoli, garlic, eggplant, pepper, pumpkin, onions, tomatoes, potatoes,sweet potatoes, cucurbits, and paprika); lauraceous plants (e.g.,avocados, cinnamon, and camphor); energy and raw material plants (e.g.,corn, soybean, rape, canola, sugar cane, and oil palm); tobacco; nuts(including peanuts); coffee; tea; vines (e.g., table grapes and juicegrape vines); hop; stone fruit; apple; blueberry; strawberry; pear;citrus; raspberry; pineapple; sugarcane; turf, natural rubber plants,and marijuana.

“Horticultural plants” are plants commonly used in horticulture andinclude, without limitation, ornamentals, vegetables, and fruits.“Ornamental” plants are plants which are commonly used in gardening,e.g., in parks, gardens, and on balconies and patios. Non-limitingexamples of ornamentals include turf, geranium, pelargonia, petunia,begonia, and fuchsia. Non-limiting examples of vegetables are asdescribed above. Non-limiting examples of fruits include apples, pears,cherries, strawberry, citrus, peaches, apricots, and blueberries.

“Silvicultural” plants are understood to be trees, more specifically,trees used in reforestation or industrial plantations. Industrialplantations generally serve the purpose of commercial production offorest products such as wood, pulp, paper, rubber tree, Christmas trees,or young trees for gardening purposes. Non-limiting examples ofsilvicultural plants are conifers (e.g., pines), in particular Pinusspecies fir and spruce; eucalyptus; tropical trees (e.g., teak, rubbertree, oil palm); willow (Salix), in particular Salix species; poplar(cottonwood), in particular Populus species; beech, in particular Fagusspecies; birch; oil palm; cherry, walnut, and oak.

In one embodiment, the plant used in the method of the present inventionis selected from dry bean, peanut, pea, and soybean.

As noted above, the term “plant” also includes plants modified fromtheir wildtype form. Such modifications may occur through breeding,mutagenesis, or genetic engineering (including transgenic andnon-transgenic plants). Plants modified by genetic engineering includeplants having genetic material that has been modified by the use ofrecombinant DNA techniques. Such modifications typically includemodifications that cannot readily be obtained by cross breeding undernatural circumstances, mutations, or natural recombination. Typically,one or more genes have been integrated into the genetic material of agenetically modified plant in order to improve certain properties of theplant. Examples of genetically modified plants are described below.

Plants used in the method of the present invention, e.g., crops whichtolerate the action of herbicides, fungicides, or insecticides owing tobreeding, including genetic engineering methods, or plants which havemodified characteristics in comparison with existing plants, which canbe generated by, e.g., traditional breeding methods and/or thegeneration of mutants, or by recombinant procedures.

In one embodiment, plants to which thiophanate methyl and an isolatedhypersensitive response elicitor protein or polypeptide fragment areapplied pursuant to the method of the present invention include plantsrendered tolerant to the application of specific classes of herbicides.Tolerance to herbicides can be obtained by creating insensitivity at thesite of action of the herbicide by expression of a target enzyme whichis resistant to herbicide, rapid metabolism (conjugation or degradation)of the herbicide by expression of enzymes which inactivate herbicide, orpoor uptake and translocation of the herbicide.

Non-limiting examples include the expression of enzymes which aretolerant to the herbicide in comparison to wild-type enzymes, such asthe expression of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS),which is tolerant to glyphosate (see e.g., “Development andCharacterization of a CP4 EPSPS-Based, Glyphosate-Tolerant Corn Event,”Heck et al., Crop Sci. 45:329-339 (2005); Funke et al., “Molecular Basisfor the Herbicide Resistance of Roundup Ready Crops,” PNAS103:13010-13015 (2006); U.S. Pat. No. 5,188,642 to Shah et al.; U.S.Pat. No. 4,940,835 to Shah et al.; U.S. Pat. No. 5,633,435 to Barry etal.; U.S. Pat. No. 5,804,425 to Barry et al.; and U.S. Pat. No.5,627,061 to Barry et al., each of which is hereby incorporated byreference in its entirety); the expression of glutamine synthase, whichis tolerant to glufosinate and bialaphos (see e.g., U.S. Pat. Nos.5,646,024 and 5,561,236 to Leemans et al., which is hereby incorporatedby reference in its entirety); and DNA constructs coding fordicamba-degrading enzymes (see e.g., for general reference, U.S. PatentApplication Publication No. 2009/0105077 to Bhatti et al. and U.S. Pat.No. 7,105,724 to Weeks et al., each of which is hereby incorporated byreference in its entirety), for dicamba resistance in bean and corn (seePCT Publication No. WO 08/051,633 to Monsanto Technology LLC, which ishereby incorporated by reference in its entirety), cotton (see U.S. Pat.No. 5,670,454 to Grossmann et al., which is hereby incorporated byreference in its entirety), pea, potato, sorghum, soybean (see U.S. Pat.No. 5,670,454 to Grossmann et al., which is hereby incorporated byreference in its entirety), sunflower, tobacco, tomato (see U.S. Pat.No. 5,670,454 to Grossmann et al., which is hereby incorporated byreference in its entirety). Gene constructs can be obtained, forexample, from microorganisms or plants, which are tolerant toherbicides, such as the Agrobacterium strain CP4 EPSPS, which isresistant to glyphosate; Streptomyces bacteria which are resistance toglufosinate; Arabidopsis, Daucus carota, Pseudomonas ssp., or Zea mayswith chimeric gene sequences coding for HDDP (see e.g., PCT PublicationNo. WO 96/38567 to Rhone-Poulenc Agrochimie and PCT Publication No. WO04/55191 to Biogemma, each of which is hereby incorporated by referencein its entirety); Arabidopsis thaliana, which is resistant to protoxinhibitors (see e.g., U.S. Patent Application Publication No.2002/0073443 to Heifetz et al., which is hereby incorporated byreference in its entirety).

Non-limiting examples of commercially available plants with tolerance toherbicides include the corn (maize) varieties Roundup Ready® Corn,Roundup Ready 2® (Monsanto), Agrisure GT®, Agrisure GT/CB/LL®, AgrisureGT/RW® Agrisure 3000GT® (Syngenta), YieldGard VT Rootworm/RR2® andYieldGard VT Triple® (Monsanto) with tolerance to glyphosate; the cornvarieties Liberty Link® (Bayer), Herculex I®, Herculex RW®, Herculex®Xtra (Dow, Pioneer), Agrisure GT/CB/LL® and Agrisure CB/LL/RW®(Syngenta) with tolerance to glufosinate; the soybean varieties RoundupReady® Soybean (Monsanto) and Optimum GAT® (DuPont, Pioneer) withtolerance to glyphosate; the cotton varieties Roundup Ready® Cotton andRoundup Ready Flex® (Monsanto) with tolerance to glyphosate; the cottonvariety FiberMax Liberty Link® (Bayer) with tolerance to glufosinate;the cotton variety BXN® (Calgene) with tolerance to bromoxynil; thecanola varieties Navigator® and Compass® (Rhone-Poulenc) with bromoxyniltolerance; the canola variety Roundup Ready® Canola (Monsanto) withglyphosate tolerance; the canola variety InVigor® (Bayer) withglufosinate tolerance; the rice variety Liberty Link® Rice (Bayer) withglufosinate tolerance and the alfalfa variety Roundup Ready Alfalfa withglyphosate tolerance.

Other plants modified with herbicide are commonly known, and includealfalfa, apple, eucalyptus, flax, grape, lentils, oil seed rape, peas,potato, rice, sugar beet, sunflower, tobacco, tomato turf grass, andwheat with tolerance to glyphosate (see e.g., U.S. Pat. Nos. 5,188,642and 4,940,835 to Shah et al. and U.S. Pat. Nos. 5,633,435, 5,804,425,and 5,627,061 to Barry et al., each of which is hereby incorporated byreference in its entirety); beans, soybean, cotton, peas, potato,sunflower, tomato, tobacco, corn, sorghum and sugarcane with toleranceto dicamba (see e.g., U.S. Patent Application Publication No.2009/0105077 to Bhatti et al.; U.S. Pat. No. 7,105,724 to Weeks et al.;and U.S. Pat. No. 5,670,454 to Grossmann et al., each of which is herebyincorporated by reference in its entirety); pepper, apple, tomato,millet, sunflower, tobacco, potato, corn, cucumber, wheat, soybean andsorghum with tolerance to 2,4-D (see e.g., U.S. Pat. Nos. 6,153,401 and6,100,446 to Streber et al.; PCT Publication No. WO 05/107437 to DowAgrosciences LLC; U.S. Pat. No. 5,608,147 to Kaphammer; and U.S. Pat.No. 5,670,454 to Grossmann et al., each of which is hereby incorporatedby reference in its entirety); sugarbeet, potato, tomato, and tobaccowith tolerance to glufosinate (see e.g. U.S. Pat. Nos. 5,646,024 and5,561,236 to Leemans et al., each of which is hereby incorporated byreference in its entirety); canola, barley, cotton, juncea, lettuce,lentils, melon, millet, oats, oilseed rape, potato, rice, rye, sorghum,soybean, sugarbeet, sunflower, tobacco, tomato, and wheat with toleranceto acetolactate synthase (ALS) inhibiting herbicides, such astriazolopyrimidine sulfonamides, growth inhibitors and imidazolinones(see e.g., U.S. Pat. No. 5,013,659 to Bedbrook et al.; PCT PublicationNo. WO 06/060634 to BASF Agrochemical Products, B.V.; U.S. Pat. Nos.4,761,373, 5,304,732, 6,211,438, 6,211,439, and 6,222,100 to Anderson etal., each of which is hereby incorporated by reference in its entirety);cereal, sugar cane, rice, corn, tobacco, soybean, cotton, rapeseed,sugar beet, and potato with tolerance to HPPD inhibitor herbicides (seee.g. PCT Publication No. WO 04/055191 to Biogemma, PCT Publication No.WO 96/38567 to Rhone-Poulenc Agrochimie, PCT Publication No. WO97/049816 to E.I. Du Pont De Nemours and Co., and U.S. Pat. No.6,791,014 to Garcon et al., each of which is hereby incorporated byreference in its entirety); wheat, soybean, cotton, sugar beet, rape,rice, corn, sorghum and sugar cane with tolerance to protoporphyrinogenoxidase (PPO) inhibitor herbicides (see e.g., U.S. Patent ApplicationPublication No. 2002/0073443 to Heifetz et al., U.S. Patent ApplicationPublication No. 2008/0052798 to Dam et al., and Li and Nicholl,“Development of PPO Inhibitor-resistant Cultures and Crops,” PestManagement Science 61:277-285 (2005), each of which is herebyincorporated by reference in its entirety). The methods of producingsuch herbicide resistant plants are generally known to persons ofordinary skill in the art, and are described in the references citedherein. Further examples of commercially available modified plants withtolerance to herbicides include CLEARFIELD® Corn, CLEARFIELD® Canola,CLEARFIELD® Rice, CLEARFIELD® Lentils, and CLEARFIELD® Sunflowers (BASF)with tolerance to the imidazolinone herbicides.

Other plants may include those able to synthesize one or moreinsecticidal proteins, especially those known from the bacterial genusBacillus, particularly from Bacillus thuringiensis, such asδ-endotoxins, e.g., CryIA(b), CryIA(c), CryIF, CryIF(a2), CryIIA(b),CryIIIA, CryIIIB(b1), and Cry9c; vegetative insecticidal proteins(“VIP”), e.g., VIP1, VIP2, VIP3, and VIP3A; insecticidal proteins ofbacteria colonizing nematodes, e.g., Photorhabdus spp. or Xenorhabdusspp.; toxins produced by animals, such as scorpion toxins, arachnidtoxins, wasp toxins, and other insect-specific neurotoxins; toxinsproduced by fungi, such Streptomycetes toxins; plant lectins, such aspea or barley lectins; agglutinins; proteinase inhibitors, such astrypsin inhibitors, serine protease inhibitors, patatin, cystatin orpapain inhibitors; ribosome-inactivating proteins (“RIP”), such asricin, corn-RIP, abrin, luffin, saporin, and bryodin; steroid metabolismenzymes, such as 3-hydroxysteroid oxidase,ecdysteroid-IDP-glycosyl-transferase, cholesterol oxidases, ecdysoneinhibitors, and HMG-CoA-reductase; ion channel blockers, such asblockers of sodium or calcium channels; juvenile hormone esterase;diuretic hormone receptors (helicokinin receptors); stilben synthase;bibenzyl synthase; chitinases; and glucanases.

The above insecticidal proteins or toxins are to be understood also aspre-toxins, hybrid proteins, truncated, or otherwise modified proteins.Hybrid proteins are characterized by a new combination of proteindomains, (see e.g., PCT Publication No. WO 02/015701 to SyngentaParticipations AG, which is hereby incorporated by reference in itsentirety). Further examples of such toxins or genetically modifiedplants capable of synthesizing such toxins are disclosed, e.g., inEuropean Patent No. 0374753 and PCT Publication Nos. WO 93/007278 and WO95/34656, all to Ciba-Geigy AG; European Patent No. 0427529 to PioneerHi Bred Int.; European Patent No. 0451878 to Bayer Bioscience NV; andPCT Publication Nos. WO 03/18810 and WO 03/52073 to SyngentaParticipations AG, each of which is hereby incorporated by reference inits entirety. Methods for producing such genetically modified plants aregenerally known to the person of ordinary skill in the art and aredescribed, e.g., in the above-cited references.

These insecticidal proteins contained in the genetically modified plantsimpart to the plants producing these proteins tolerance to harmful pestsfrom all taxonomic groups of arthropods, especially to beetles(Coleoptera), two-winged insects (Diptera), and moths (Lepidoptera) andto nematodes (Nematoda). Genetically modified plants capable tosynthesize one or more insecticidal proteins are, e.g., described in thecitations mentioned above, some of which are commercially available suchas YieldGard® (corn cultivars producing the Cry1Ab toxin), YieldGard®Plus (corn cultivars producing Cry1Ab and Cry3Bb1 toxins), Starlink®(corn cultivars producing the Cry9c toxin), Herculex® RW (corn cultivarsproducing Cry34Ab1, Cry35Ab1 and the enzymePhosphinothricin-N-Acetyltransferase [PAT]); NuCOTN® 33B (cottoncultivars producing the Cry1Ac toxin), Bollgard® I (cotton cultivarsproducing the Cry1Ac toxin), Bollgard® II (cotton cultivars producingCry1Ac and Cry2Ab2 toxins); VIPCOT® (cotton cultivars producing aVIP-toxin); NewLeaf (potato cultivars producing the Cry3A toxin);Bt-Xtra®, NatureGard®, KnockOut®, BiteGard®, Protecta®, Bt11 (e.g.,Agrisure® CB), and Bt176 from Syngenta Seeds SAS, France (corn cultivarsproducing the Cry1Ab toxin and PAT enzyme), MIR604 from Syngenta SeedsSAS, France (corn cultivars producing a modified version of the Cry3Atoxin (see PCT Publication No. WO 03/018810 to Syngenta ParticipationsAG, which is hereby incorporated by reference in its entirety), MON 863from Monsanto Europe S.A., Belgium (corn cultivars producing the Cry3Bb1toxin), IPC531 from Monsanto Europe S.A., Belgium (cotton cultivarsproducing a modified version of the Cry1Ac toxin), and 1507 from PioneerOverseas Corporation, Belgium (corn cultivars producing the Cry1F toxinand PAT enzyme).

Plants also include those that, through the use of recombinant DNAtechniques, are able to synthesize one or more proteins to increase theresistance or tolerance of those plants to bacterial, viral, or fungalpathogens. Non-limiting examples of such proteins are the so-called“pathogenesis-related proteins” (“PR” proteins, see e.g., EuropeanPatent No. 0392225 to Syngenta Participations AG, which is herebyincorporated by reference in its entirety), plant disease resistancegenes (e.g., potato cultivars, which express resistance genes actingagainst Phytophthora infestans derived from the Mexican wild potatoSolanum bulbocastanum) or T4-lysozyme (e.g., potato cultivars capable ofsynthesizing these proteins with increased resistance against bacteriasuch as Erwinia amylovora). The methods for producing such geneticallymodified plants are generally known to the person of ordinary skill inthe art and are described, e.g., in the above-noted references.

Plants amenable to the method of the present invention also includethose that through the use of recombinant DNA techniques are capable ofsynthesizing one or more proteins to increase the productivity (e.g.,biomass production, grain yield, starch content, oil content, and/orprotein content), tolerance to drought, salinity, or othergrowth-limiting environmental factors or tolerance to pests and fungal,bacterial, or viral pathogens of those plants.

Suitable plants also include those that through the use of recombinantDNA techniques contain a modified amount of substances of content or newsubstances of content, specifically to improve human or animalnutrition, e.g., oil crops that produce health-promoting long-chainomega-3 fatty acids or unsaturated omega-9 fatty acids (e.g., Nexera®rape, DOW Agro Sciences, Canada).

Suitable plants also include those that through the use of recombinantDNA techniques contain a modified amount of substances of content or newsubstances of content, specifically to improve raw material production,e.g., potatoes that produce increased amounts of amylopectin (e.g.,Amflora® potato, BASF SE, Germany).

Particularly suited plants have been rendered tolerant to at least oneherbicide, are resistant to glyphosate or an agriculturally acceptablesalt thereof, and/or are resistant to dicamba or an agriculturallyacceptable salt thereof.

The method of the present invention involves applying thiophanate methyland an isolated hypersensitive response elicitor protein or polypeptidefragment to a plant and/or area of cultivation.

Thiophanate methyl is a fungicide which belongs to the functional classof benzimidazole precursor fungicides, or thiophanates. These fungicidesact by disrupting β-tubulin assembly in mitosis. Based on the results inthe accompanying examples, it is believed similar synergistic effectscan be achieved with other members of this class of benzimidazoles.

Isolated HR elicitor proteins or polypeptide fragments are known in theart. In one embodiment, the isolated HR elicitor protein or polypeptidefragment is a full length HR elicitor protein, also known as harpin.Harpin includes any member of the art-recognized class of proteins thatare produced by plant bacteria, and which share structural features anda capacity for inducing a plant hypersensitive response. Biochemically,these proteins or polypeptides have a number of common structuralcharacteristics. These include being glycine rich, low or no cysteinecontent, heat stable, hydrophilic, lacking an N-terminal signalsequence, and susceptible to proteolysis. See Bonas, “Bacterial HomeGoal by Harpins,” Trends Microbiol. 2:1-2 (1994); Gopalan et al.,“Bacterial Genes Involved in the Elicitation of Hypersensitive Responseand Pathogenesis,” Plant Disease 80:604-10 (1996); and Alfano et al.,“The Type III (Hrp) Secretion Pathway of Plant Pathogenic Bacteria:Trafficking Harpins, Avr Proteins, and Death,” Journal of Bacteriology179:5655-5662 (1997), each of which is hereby incorporated by referencein its entirety. In addition, harpins share a unique secondary structurethat has been associated with their distinct biological activities. Thestructure has two primary components, an alpha helix unit and a relaxedacidic unit having a sheet or random turn structure. In the absence ofone or both of these components, hypersensitive response elicitationdoes not occur. See PCT Publication No. WO 01/98501 to Fan et al., whichis hereby incorporated by reference in its entirety.

The harpin proteins also share the ability to induce specific plantresponses (i.e., following their application to a plant and/or area ofcultivation). The induction of plant disease resistance, plant growth,insect resistance, desiccation resistance, and post-harvest diseaseresistance (in harvested plant products, such as fruits and vegetables)are several of the more important utilities. These uses of the harpinproteins are described in U.S. Pat. No. 6,277,814 to Qiu et al.; U.S.Pat. No. 5,776,889 to Wei et al.; U.S. Pat. No. 5,977,060 to Zitter etal.; U.S. Pat. No. 6,235,974 to Qiu et al.; U.S. Patent ApplicationPublication No. 2003/0104979 to Wei et al.; U.S. Patent ApplicationPublication No. 2002/0019337 to Wei et al.; and U.S. Patent ApplicationPublication No. 2004/0265442; each of which is hereby incorporated byreference in its entirety. The induction of these responses is due toupregulation of jasmonic acid/ethylene and salicylic acid defensepathways, as well as plant growth pathways that regulate photosynthesisand nutrient uptake.

One group of harpin proteins or polypeptides includes, withoutlimitation, homologs of Erwinia amylovora HrpN, which include those fromspecies of Erwinia, Pantoea, and Pectobacterium. Examples of suchhomologs include those harpin proteins identified at Genbank AccessionNos. AAC31644 (Erwinia amylovora); AAQ21220, AAQ17045, CAE25423,CAE25424, CAE25425, and CAF74881 (Erwinia pyrifoliae); CAC20124, Q47278,Q47279, and AAY17519 (Erwinia chrysanthemi); CAE25422 (Erwinia strainJP557); AAG01466 (Pantoea stewartii); AAF76342 (Pantoea agglomerans);ABZ05760, ABI15988, ABI15989, ABI15990, ABI15991, ABI15992, ABI15996,ABK80762, ABD04037, ABI15994, ABD04035, ABD04036, AAY17521, AAX38231,ABI15995, AAQ73910, and CAL69276 (Pectobacterium carotovorum);YP_(—)050198, AAS20361, and CAE45180 (Pectobacterium atrosepticum); andABD22989 (Pectobacterium betavasculorum), each of which is herebyincorporated by reference in its entirety.

Another group of harpin proteins or polypeptides includes, withoutlimitation, homologs of Erwinia amylovora HrpW and Pseudomonas syringaeHrpW, which includes those from species of Erwinia, Pseudomonas,Xanthomonas, Acidovorax, and Pectobacterium. Examples of such homologsinclude those harpin proteins identified at Genbank Accession Nos.U94513, CAA74158, AAC04849, and AAF63402 (Erwinia amylovora); AAQ17046(Erwinia pyrifoliae); YP_(—)001906489 (Erwinia tasmaniensis);YP_(—)050207 (Pectobacterium atrosepticum); AF037983 (Pseudomonassyringae pv. tomato); AA050075 (Pseudomonas syringae pv. phaseolicola);AAL84244 (Pseudomonas syringae pv. maculicola); AAX58537, AAX58557,AAX58525, AAX58531, AAX58527, AAX58577, AAX58491, AAX58515, AAX58517,AAX58523, AAX58583, AAX58451, AAX58561, AAX58453, AAX58541, AAX58589,AAT96311, AAX58497, AAX58579, AAX58449, AAX58485, AAX58563, AAX58581,AAX58575, AAX58569, AAX58567, AAX58505, AAX58591, AAX58503, AAX58507,AAX58509, AAX58469, AAX58441, AAX58543, AAX58495, AAX58549, AAX58593,AAX58511, AAX58519, AAT96270, AAX58447, AAX58571, AAX58465, AAX58489,AAX58533, AAX58535, AAX58461, AAT96350, AAX58473, AAX58483, AAX58475,AAX58457, AAX52461, AAX52457, AAT96222, (Pseudomonas viridiflava);ABA47299 and BAG24117 (Pseudomonas cichorii); CAH57075 (Pseudomonasavellanae); BAE80274 and BAE80242 (Acidovorax avenae); and AAM37767(Xanthomonas axonopodis pv. citri), each of which is hereby incorporatedby reference in its entirety.

Yet another group of harpin proteins or polypeptides includes, withoutlimitation, homologs of Pseudomonas syringae HrpZ, which includes thosefrom other species of Pseudomonas. Examples of such homologs includethose harpin proteins identified at Genbank Accession Nos. P35674,AABO0127, ABL01505, AAQ92359, BAD20880, BAD20876, BAD20892, BAD20884,BAD20928, BAD20936, BAD20932, BAD20924, BAD20856, BAD20864, BAD20860,BAD20848, BAD20844, BAD20836, BAD20840, BAD20824, BAD20842, BAD20820,BAD20916, BAD20872, BAC81526, O87653, BAA74798, BAD20904, AAB86735,BAD20912, BAD20908, ABL01504, BAB40655, ABO26225, ABO26228 (Pseudomonassyringae pv.); BAD20868 (Pseudomonas ficuserectae); AAX52452, AAT96159,AAX52266, AAX52396, AAT96322, AAT96281, AAX52272, AAX52306, AAX52270,AAX52402, AAX52276, AAX52318, AAX52262, and AAT96361 (Pseudomonasviridiflava); CAJ76697 (Pseudomonas avellanae); YP_(—)001185537(Pseudomonas mendocina); and ABA47309 and BAG24128 (Pseudomonascichorii), each of which is hereby incorporated by reference in itsentirety.

An additional group of harpin proteins or polypeptides includes, withoutlimitation, homologs of Xanthomonas campestris HreX (see U.S. Pat. No.6,960,705 to Wei et al., which is hereby incorporated by reference inits entirety), which includes those from other species of Xanthomonas.Non-limiting examples of such homologs include those harpin proteinsidentified at Genbank Accession Nos. NP_(—)636614, YP_(—)001904470,YP_(—)362171 (Xanthomonas campestris); ABB72197, ABK51585, ABU48601,ABK51584, YP_(—)198734, and ZP_(—)02245223 (Xanthomonas oryzae); andABK51588 and NP_(—)640771 (Xanthomonas axonopodis); each of which ishereby incorporated by reference in its entirety.

In another embodiment, the isolated HR elicitor protein or polypeptidefragment is a fragment or combination of fragments (i.e., a fusionprotein) of one of the above referenced harpin proteins. In oneembodiment, the fragment or fusion protein includes fragments thatelicit the HR. In another embodiment, the fragment or fusion proteinincludes fragments that do not elicit the HR. Suitable fragmentsinclude, e.g., two structural units: a stable α-helix unit with 12 ormore amino acids in length; and a hydrophilic, acidic unit with 12 ormore amino acids in length, which could be a beta-form, a beta-turn, orunordered forms. Fragments may also be characterized by an acidic pIvalue that is preferably about 5 or below. Fragments may contain anynumber of amino acids, e.g., between about 25 and about 60, or betweenabout 28 to about 40 amino acids.

Examples of suitable fragments are identified in U.S. Pat. No. 6,583,107to Laby et al., and PCT Publication No. WO 01/098501 to Fan et al., eachof which is hereby incorporated by reference in its entirety. PCTPublication No. WO 01/098501 to Fan et al. also describes methods forobtaining fragments of harpin protein or polypeptides that could beemployed in the present invention.

Suitable HR-eliciting polypeptide fragments include, without limitation,those identified in Table 1.

TABLE 1 List of HR-Eliciting Fragments HR domain Isolated Source AminoAcid Residues pI HrpN_(Ea)-1 E. amylovora 43-70 3.09 HrpN_(Ea)-2 E.amylovora 140-176 3.17 HrpN_(Ech)-1 E. chrysanthemi  78-118 5.25HrpN_(Ech)-2 E. chrysanthemi 256-295 4.62 HrpN_(Ecc)-1 E. carotovora25-63 4.06 HrpN_(Ecc)-2 E. carotovora 101-140 3.00 HrpW_(Pss)-1 P.syringae 52-96 4.32 HrpW_(Ea)-1 E. amylovora 10-59 4.53 HrpZ_(Pss)-1 P.syringae  97-132 3.68 HrpZ_(Pss)-2 P. syringae 153-189 3.67 HrpZ_(Pss)-3P. syringae 271-308 3.95 PopA1_(Rs)-1 R. solanacearum  92-125 3.75PopA1_(Rs)-2 R. solanacearum 206-260 3.62

As noted above, suitable fragments of harpin protein or polypeptides maynot elicit the hypersensitive response in plants, but may still beuseful in the method and compositions of the present invention. Suchfragments are described in U.S. Pat. No. 6,858,707 to Wei et al., whichis hereby incorporated by reference in its entirety.

Examples of suitable fragments of a hypersensitive response elicitorwhich do not elicit a hypersensitive response include fragments of theErwinia amylovora hypersensitive response elicitor are described in U.S.Pat. No. 6,858,707 to Wei et al., which is hereby incorporated byreference in its entirety. These include the C-terminal fragments of theHrpN amino acid sequence recited in U.S. Pat. No. 6,858,707 (which ishereby incorporated by reference in its entirety) that span the aminoacids 169-403, 210-403, 267-403, or 343-403; and internal fragments ofthe HrpN amino acid sequence recited in U.S. Pat. No. 6,858,707 (whichis hereby incorporated by reference in its entirety) that span the aminoacids 150-179, 137-166, 121-150, 76-168, 105-168, or 137-156.

Another example of a useful fragment of a hypersensitive responseelicitor which fragment does not itself elicit a hypersensitive responseis the protein fragment containing amino acids 190 to 294 of the HrpZamino acid sequence as recited in U.S. Pat. No. 6,858,707 (which ishereby incorporated by reference in its entirety).

Variants of fragments of hypersensitive response elicitors that do notelicit a hypersensitive response may be made by, for example, thedeletion or addition of amino acids that have minimal influence on theproperties, secondary structure, and hydropathic nature of thepolypeptide. For example, a polypeptide may be conjugated to a signal(or leader) sequence at the N-terminal end of the protein whichco-translationally or post-translationally directs transfer of theprotein. The polypeptide may also be conjugated to a linker or othersequence for ease of synthesis, purification, or identification of thepolypeptide.

Suitable fragments can be produced by several means. According to oneapproach, subclones of the gene encoding a known harpin protein orpolypeptide are produced by conventional molecular genetic manipulationby subcloning gene fragments. The subclones then are expressed in vitroor in vivo in bacterial cells to yield a smaller protein or peptide thatcan be tested for activity.

As an alternative approach, fragments can be produced by digestion of afull-length harpin protein or polypeptide with proteolytic enzymes likechymotrypsin or Staphylococcus proteinase A, or trypsin. Differentproteolytic enzymes are likely to cleave elicitor proteins at differentsites based on the amino acid sequence of the harpin protein. Some ofthe fragments that result from proteolysis may be active elicitors.

In yet another approach, based on knowledge of the primary structure ofthe protein, fragments of the harpin protein gene may be synthesized byusing the PCR technique together with specific sets of primers chosen torepresent particular portions of the protein. These then would be clonedinto an appropriate vector for expression of a truncated peptide orprotein.

Chemical synthesis can also be used to make suitable fragments. Such asynthesis is carried out using known amino acid sequences for the harpinbeing produced. Alternatively, subjecting a full length harpin to hightemperatures and pressures will produce fragments. These fragments canthen be separated by conventional procedures (e.g., chromatography,SDS-PAGE).

Harpin protein or polypeptides of the present invention may also includeisolated hypersensitive response elicitor fusion proteins comprising twoor more spaced apart HR-eliciting or non-eliciting domains.

Building blocks containing one or more HR-eliciting domains include,without limitation, the building blocks identified in Table 2.

TABLE 2 Building Block Domains for Fusion Proteins Domain SequenceSource MW (kDa) # a.a. pI A PopA70-146 10.69 104 6.48 (N_(N))HrpN_(Ea)40-80 6.754 68 6.78 (N_(N))₂ Dimer of HrpN_(Ea)40-80 10.84 1116.13 (N_(N))₃ Triplemer of HrpN_(Ea)40-80 14.93 154 5.63 (N_(N))₄Tetramer of HrpN_(Ea)40-80 19.01 197 4.95 (N_(C)) HrpN_(Ea)140-180 7.22468 5.01 (N_(C))₂ Dimer of HrpN_(Ea)140-180 11.78 111 3.98 (N_(C))₃Trimer of HrpN_(Ea)140-180 16.34 154 3.72 (N_(C))₄ Tetramer ofHrpN_(Ea)140-180 20.89 197 3.58 (N_(C))₁₀ Decamer of HrpN_(Ea)140-18048.23 455 3.28 (N_(C))₁₆ Hexadecamer of HrpN_(Ea)140-180 75.57 713 3.18W HrpW_(Ea)10-59 7.986 77 6.48 Z_(N) HrpZ90-150 8.087 78 5.38 Z₂₆₆₋₃₀₈HrpZ266-308 7.029 70 6.40

With the combination of these (and other) HR-eliciting domains, fusionproteins can be produced that have higher HR potency and have enhancedability to induce desired plant response, in this case increased yield,and enhanced growth. These fusion proteins can be formed using one HRdomain repeat unit (concatomer), different combinations of HR domains,and/or biologically active domains from other elicitors.

Using these building blocks, several isolated fusion proteins include,without limitation, those identified in Table 3.

TABLE 3 Fusion Protein Constructions MW Protein Domain Sequence (kDa) #a.a. pI SH-1 *W(N_(N))₄A(N_(C))₄ 54.955 545 3.69 Z₂₆₆₋₃₀₈ SH-2*W(N_(N))₄Z_(N)(N_(C))₄ 52.341 519 3.54 Z₂₆₆₋₃₀₈ SH-3*W(N_(N))₄Z_(N)(N_(C))₄ 60.375 598 3.67 Z₂₆₆₋₃₀₈A

These fusion proteins are heat stable and soluble, and have beendemonstrated to possess improved growth enhancement activity as comparedto harpin proteins isolated from plant pathogenic bacteria, such asHrpN. These fusion proteins are described in PCT Publication No. WO01/098501 to Fan et al., which is hereby incorporated by reference inits entirety.

One preferred fusion protein, now commercially available from PlantHealth Care Inc., is characterized by the amino acid sequence of SEQ IDNO:1 as follows:

MSLNTSGLGA STMQISIGGA GGNNGLLGTH MPGTSSSPGLFQSGGDNGLG GHNANSALGQ QPIDRQTIEQ MAQLLAELLKSLLDSGEKLG DNFGASADSA SGTGQQDLMT QVLNGLAKSMLDDLLTKQDG GTSFSEDDSG PAKDGNANAG ANDPSKNDPSKSQGPQSANK TGNVDDANNQ DPMQALMQLL EDLVKLLKAALHMQQPGGND KGNGVGGDSG QNDDSTSGTD STSDSSDPMQ QLLKMFSEIM QSLFGDEQDG TDSTSGSRFT RTGIGMKAGIQALNDIGTHS DSSTRSFVNK GDRAMAKEIG QFMDQYPEVFGKPQYQKGPG QEVKTDDKSW AKALSKPDDD GMTPASMEQFNKAKGMIKSA MAGDTGNGNL QARGAGGSSL GIDAMMAGDA INNMALGKLG AA

Residues 1-30 of SEQ ID NO:1 correspond to the N-terminal sequence ofHrpN_(Ea); residues 31-34 (bold) are artifacts of ligating the HRdomains together; residues 35-83 correspond to one HR domain ofHrpW_(Ea) (residues 10-59); residues 84-86 (bold) are artifacts ofligating the HR domains together; residues 87-138 correspond to one HRdomain of HrpZ_(Pss) (residues 90-141); residues 139-140 (bold) areartifacts of ligating the HR domains together; residues 141-211correspond to one HR domain of PopA (residues 70-140); residues 212-220correspond to artifacts of ligating the HR domains together; residues221-261 correspond to one HR domain of HrpN_(Ea) (residues 140-180);residues 262-271 correspond to artifacts of ligating the HR domainstogether; and residues 272-412 correspond to the C-terminal sequence ofHrpN_(Ea) (residues 263-403).

The fusion protein of SEQ ID NO:1 is encoded by the nucleotide sequenceof SEQ ID NO:2 as follows:

ATGAGTCTGA ATACAAGTGG GCTGGGAGCG TCAACGATGCAAATTTCTAT CGGCGGTGCG GGCGGAAATA ACGGGTTGCTGGGTACGCAT ATGCCCGGGA CCTCGTCCTC GCCGGGTCTGTTCCAGTCCG GGGGGGACAA CGGGCTTGGT GGTCATAATGCAAATTCTGC GTTGGGGCAA CAACCCATCG ATCGGCAAACCATTGAGCAA ATGGCTCAAT TATTGGCGGA ACTGTTAAAGTCACTGCTAG ATAGTGGGGA AAAGCTCGGT GACAACTTCGGCGCGTCTGC GGACAGCGCC TCGGGTACCG GACAGCAGGACCTGATGACT CAGGTGCTCA ATGGCCTGGC CAAGTCGATGCTCGATGATC TTCTGACCAA GCAGGATGGC GGGACCAGCTTCTCCGAAGA CGATAGTGGG CCGGCGAAGG ACGGCAATGCCAACGCGGGC GCCAACGACC CGAGCAAGAA CGACCCGAGCAAGAGCCAGG GTCCGCAGTC GGCCAACAAG ACCGGCAACGTCGACGACGC CAACAACCAG GATCCGATGC AAGCGCTGATGCAGCTGCTG GAAGACCTGG TGAAGCTGCT GAAGGCGGCCCTGCACATGC AGCAGCCCGG CGGCAATGAC AAGGGCAACGGCGTGGGCGG TGATAGTGGG CAAAACGACG ATTCCACCTCCGGCACAGAT TCCACCTCAG ACTCCAGCGA CCCGATGCAGCAGCTGCTGA AGATGTTCAG CGAGATAATG CAAAGCCTGTTTGGTGATGA GCAAGATGGC ACCGATAGTA CTAGCGGCTCGAGGTTTACT CGTACCGGTA TCGGTATGAA AGCGGGCATTCAGGCGCTGA ATGATATCGG TACGCACAGC GACAGTTCAACCCGTTCTTT CGTCAATAAA GGCGATCGGG CGATGGCGAAGGAAATCGGT CAGTTCATGG ACCAGTATCC TGAGGTGTTTGGCAAGCCGC AGTACCAGAA AGGCCCGGGT CAGGAGGTGAAAACCGATGA CAAATCATGG GCAAAAGCAC TGAGCAAGCCAGATGACGAC GGAATGACAC CAGCCAGTAT GGAGCAGTTCAACAAAGCCA AGGGCATGAT CAAAAGCGCC ATGGCGGGTGATACCGGCAA CGGCAACCTG CAGGCACGCG GTGCCGGTGGTTCTTCGCTG GGTATTGATG CCATGATGGC CGGTGATGCCATTAACAATA TGGCACTTGG CAAGCTGGGC GCGGCTTAA

In carrying out the method of the present invention, application of thefungicide thiophanate methyl and the isolated hypersensitive responseelicitor protein or polypeptide fragment may occur by either applying tothe plant and/or area of cultivation each compound in sequence or byapplying a single composition containing both compounds. Either way, theapplication of both compounds (either sequentially or simultaneously) iscarried out to synergistically increase yield of the plant.

In one embodiment, application is carried out by applying a formulationof thiophanate methyl and a formulation of an isolated hypersensitiveresponse elicitor protein or polypeptide fragment, either at the sametime (e.g., combined into a single formulation mixture, or appliedsimultaneously as two individual formulations) or in succession toachieve substantially the same result.

Application in succession means thiophanate methyl is applied before orafter an isolated HR elicitor protein or polypeptide fragment. Whenapplication of the two compounds occurs successively, the time intervalbetween the individual applications is selected to ensure that theactive substance applied first still occurs at the site of action in asufficient amount at the time of application of the further activesubstance(s). Preferably, the time interval for a subsequent applicationof an active compound (and any additional compounds) ranges from about afew seconds up to about 3 months, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, orabout 10 seconds, 1 minute, about 1-3, 5, 10, 15, 30, or 45 minutes,about 1 hour, 2-3 hours, 5 hours, 10 hours, 15 hours, 20 hours, or 24hours, about 1-3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1month, or about 1-5 months.

The order of application of the two substances is not essential foroperation of the present invention. Furthermore, as discussed more fullybelow, application of one or both of these substances or theircombination, can be repeated one or more times during the growingseason.

Thiophanate methyl and isolated HR elicitor protein or polypeptidefragments may be formulated into two separate formulations orcompositions, or a single formulation or composition.

Agricultural formulations of active substances are well known.Non-limiting examples include solutions, emulsions, suspensions, dusts,powders, pastes, and granules. The particular formulation chosen mayvary depending on the particular intended application. In each case, itis typically advantages to ensure a fine and even distribution of theactive ingredient(s) in a liquid or solid carrier.

Formulation methods are taught, e.g., in U.S. Pat. No. 3,060,084 toLittler and European Patent No. 0707445 to BASF AG (for liquidconcentrates); Browning, “Agglomeration,” Chemical Engineering pp.147-48 (1967); Perry's Chemical Engineer's Handbook, 4th Ed.,McGraw-Hill, New York, 1963; PCT Publication No. WO 91/13546 to E.I. DuPont De Nemours and Co.; U.S. Pat. No. 4,172,714 to Albert; U.S. Pat.No. 4,144,050 to Frensch et al.; U.S. Pat. No. 3,920,442 to Albert; U.S.Pat. No. 5,180,587 to Moore; U.S. Pat. No. 5,232,701 to Ogawa et al.;U.S. Pat. No. 5,208,030 to Hoy et al., Great Britain Patent No.2,095,558; U.S. Pat. No. 3,299,566 to Macmullen; Klingman, Weed Controlas a Science, J. Wiley & Sons, New York, 1961; Hance et al., WeedControl Handbook, 8th Ed., Blackwell Scientific, Oxford, 1989; andMollet and Grubemann, Formulation Technology, Wiley VCH Verlag,Weinheim, 2001, each of which is hereby incorporated by reference in itsentirety.

Thiophanate methyl and isolated HR elicitor protein or polypeptidefragments may be formulated (either together or separately) in a mannercommon for agrochemical formulations. For example, the composition(s)may include auxiliaries which are customary in agrochemicalformulations. The particular auxiliaries used may depend on theparticular application form and active substance, respectively.Non-limiting examples of suitable auxiliaries include solvents, solidcarriers, dispersants, or emulsifiers (e.g., further solubilizers,protective colloids, surfactants, and adhesion agents), organic andinorganic thickeners, bactericides, anti-freezing agents, anti-foamingagents, colorants, tackifiers or binders, or combinations thereof.

Suitable solvents include water; organic solvents such as mineral oilfractions of medium to high boiling point, coal tar oils and oils ofvegetable or animal origin; aliphatic, cyclic and aromatic hydrocarbons(e.g., toluene, xylene, paraffin, tetrahydronaphthalene, alkylatednaphthalenes, or their derivatives); alcohols such as methanol, ethanol,propanol, butanol, and cyclohexanol; glycols; ketones such ascyclohexanone and gamma-butyrolactone; fatty acid dimethylamides; fattyacids and fatty acid esters; and strongly polar solvents (e.g., aminessuch as N-methylpyrrolidone).

Suitable solid carriers include mineral earths such as silicates, silicagels, talc, kaolins, limestone, lime, chalk, bole, loess, clays,dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate,magnesium oxide, ground synthetic materials, fertilizers (e.g., ammoniumsulfate, ammonium phosphate, ammonium nitrate, ureas, and products ofvegetable origin, such as cereal meal, tree bark meal, wood meal andnutshell meal); cellulose powders; and other solid carriers.

Suitable surfactants (also known as adjuvants, wetters, tackifiers,dispersants, or emulsifiers) are alkali metal, alkaline earth metal, andammonium salts of aromatic sulfonic acids (e.g., ligninsulfonic acid(Borresperse® types, Borregard, Norway)) phenolsulfonic acid,naphthalenesulfonic acid (Morwet® types, Akzo Nobel, U.S.A.),dibutylnaphthalene-sulfonic acid (Nekal® types, BASF, Germany), fattyacids, alkylsulfonates, alkylarylsulfonates, alkyl sulfates, laurylethersulfates, fatty alcohol sulfates, sulfated hexa-, hepta- andoctadecanolates, sulfated fatty alcohol glycol ethers, condensates ofnaphthalene or of naphthalenesulfonic acid with phenol and formaldehyde,polyoxy-ethylene octylphenyl ether, ethoxylated isooctylphenol,octylphenol, nonylphenol, alkylphenyl polyglycol ethers, tributylphenylpolyglycol ether, tristearylphenyl polyglycol ether, alkylaryl polyetheralcohols, alcohol and fatty alcohol/ethylene oxide condensates,ethoxylated castor oil, polyoxyethylene alkyl ethers, ethoxylatedpolyoxypropylene, lauryl alcohol polyglycol ether acetal, sorbitolesters, lignin-sulfite waste liquid and proteins, denatured proteins,polysaccharides (e.g., methylcellulose), hydrophobically modifiedstarches, polyvinyl alcohols (Mowiol® types, Clariant, Switzerland),polycarboxylates (Sokolan® types, BASF, Germany), polyalkoxylates,polyvinylamines (Lupasol® types, BASF, Germany), polyvinylpyrrolidoneand copolymers thereof.

Examples of thickeners (i.e., compounds that impart a modifiedflowability to formulations (i.e., high viscosity under staticconditions and low viscosity during agitation) are polysaccharides andorganic and inorganic clays such as Xanthan gum (Kelzan®, CP Kelco,U.S.A.), Rhodopol® 23 (Rhodia, France), Veegum® (R.T. Vanderbilt,U.S.A.) or Attaclay® (Engelhard Corp., NJ, USA).

Bactericides may be added to the composition for preservation andstabilization. Examples of suitable bactericides include, e.g., thosebased on dichlorophene and benzylalcohol hemi formal (Proxel® from ICI,Acticide® RS from Thor Chemie, and Kathon® MK from Rohm & Haas) andisothiazolinone derivatives such as alkylisothiazolinones andbenzisothiazolinones (Acticide® MBS from Thor Chemie).

Examples of suitable anti-freezing agents are ethylene glycol, propyleneglycol, urea, and glycerin.

Examples of anti-foaming agents are silicone emulsions (e.g., Silikon®SRE, Wacker, Germany and Rhodorsil®, Rhodia, France), long chainalcohols, fatty acids, salts of fatty acids, fluoroorganic compounds,and mixtures thereof.

Suitable colorants are pigments of low water solubility andwater-soluble dyes. Non-limiting examples include rhodamin B, C. I.pigment red 112, C. I. solvent red 1, pigment blue 15:4, pigment blue15:3, pigment blue 15:2, pigment blue 15:1, pigment blue 80, pigmentyellow 1, pigment yellow 13, pigment red 112, pigment red 48:2, pigmentred 48:1, pigment red 57:1, pigment red 53:1, pigment orange 43, pigmentorange 34, pigment orange 5, pigment green 36, pigment green 7, pigmentwhite 6, pigment brown 25, basic violet 10, basic violet 49, acid red51, acid red 52, acid red 14, acid blue 9, acid yellow 23, basic red 10,and basic red 108.

Examples of tackifiers or binders include polyvinylpyrrolidones,polyvinylacetates, polyvinyl alcohols, and cellulose ethers (e.g.,Tylose®, Shin-Etsu, Japan).

Powders, materials for spreading, and dusts can be prepared by mixing orconcomitantly grinding the active compounds with at least one solidcarrier.

Granules (e.g., coated granules, impregnated granules, and homogeneousgranules) can be prepared by binding the active substances to solidcarriers. Examples of solid carriers are mineral earths such as silicagels, silicates, talc, kaolin, attaclay, limestone, lime, chalk, bole,loess, clay, dolomite, diatomaceous earth, calcium sulfate, magnesiumsulfate, magnesium oxide, ground synthetic materials, fertilizers (e.g.,ammonium sulfate, ammonium phosphate, ammonium nitrate, ureas, andproducts of vegetable origin, such as cereal meal, tree bark meal, woodmeal and nutshell meal), cellulose powders, and other solid carriers.

In one embodiment, the composition is formulated for dilution withwater. Thus, the composition may be formulated as a water solubleconcentrate, a dispersable concentrate, an emulsifiable concentrate, anemulsion, a suspension, water-dispersible granules and/or water-solublegranules, water-dispersible powders and/or water-soluble powders, or agel.

In the case of water-soluble concentrates, the formulation may includee.g., about 10 parts by weight of the composition of the presentinvention dissolved in about 90 parts by weight of water or awater-soluble solvent. As an alternative, wetting agents or otherauxiliaries may be added. The active substances dissolve upon dilutionwith water. In this way, a formulation having a content of about 10% byweight of active substance is obtained.

In the case of dispersible concentrates, the formulation may include,e.g., about 20 parts by weight of the active compound(s) dissolved inabout 70 parts by weight of cyclohexanone with addition of about 10parts by weight of a dispersant, e.g., polyvinylpyrrolidone. Dilutionwith water gives a dispersion. According to this formulation, the activesubstance content is about 20% by weight.

In the case of emulsifiable concentrates, the formulation may include,e.g., about 15 parts by weight of the active compound(s) dissolved inabout 75 parts by weight of xylene with addition of calciumdodecylbenzenesulfonate and castor oil ethoxylate (in each case about 5parts by weight). Dilution with water gives an emulsion. The compositionhas an active substance content of about 15% by weight.

In the case of an emulsion, the formulation may include, e.g., about 25parts by weight of active compound(s) dissolved in about 35 parts byweight of xylene with addition of calcium dodecylbenzenesulfonate andcastor oil ethoxylate (in each case about 5 parts by weight). Thismixture is introduced into about 30 parts by weight of water by means ofan emulsifying machine (Ultraturrax) and made into a homogeneousemulsion. Dilution with water gives an emulsion. The composition has anactive substance content of about 25% by weight.

In the case of a suspension, in an agitated ball mill about 20 parts byweight of the active compound(s) comminuted with an addition of about 10parts by weight of dispersants and wetting agents and about 70 parts byweight of water or an organic solvent to give a fine active substancesuspension. Dilution with water gives a stable suspension of the activesubstance. The active compound(s) in the composition is about 20% byweight.

In the case of water-dispersible granules and/or water-soluble granules,the formulation may include, e.g., about 50 parts by weight of theactive compound(s) ground finely with about 50 parts by weight ofdispersants and wetting agents and prepared as water-dispersible orwater-soluble granules by means of technical appliances (e.g.,extrusion, spray tower, fluidized bed). Dilution with water gives astable dispersion or solution of the active substance. The compositionhas an active compound(s) content of about 50% by weight.

In the case of water-dispersible powders and/or water-soluble powders,the formulation may include, e.g., about 75 parts by weight of theactive compound(s) ground in a rotor-stator mill with addition of about25 parts by weight of dispersants, wetting agents, and silica gel.Dilution with water gives a stable dispersion or solution of the activesubstances. The active compound(s) content of the composition is about75% by weight.

In the case of a gel, the formulation may include, e.g., about 20 partsby weight of the active compound(s) agitated in a ball mill andcomminuted with the addition of about 10 parts by weight of dispersants,about 1 part by weight of a gelling agent, and about 70 parts by weightof water or an organic solvent to give a fine suspension of the activesubstances. Dilution with water gives a stable suspension of the activesubstances, whereby a composition with about 20% (w/w) of activecompound(s) is obtained.

In another embodiment, the active compound(s) is formulated for anundiluted application. Thus, the active compound(s) may be formulated asa dustable powder, granules, or Ultra Low Volume (“ULV”) (e.g., fogging)solutions.

In the case of a dustable powder, the formulation may include, e.g.,about 5 parts by weight of active compound(s) ground finely and mixedintimately with about 95 parts by weight of finely divided kaolin. Thisgives a dustable composition having an active substance content of about5% by weight.

In the case of granules, the formulation may include, e.g., about 0.5parts by weight of the active compound(s) ground finely and associatedwith about 99.5 parts by weight of carriers. This gives granules to beapplied undiluted having an active compound(s) content of about 0.5% byweight.

In the case of ULV solutions, the formulation may include, e.g., about10 parts by weight of the active compound(s) dissolved in about 90 partsby weight of an organic solvent, e.g., xylene. This gives a compositionto be applied undiluted having an active compound(s) content of about10% by weight.

In the present invention, agrochemical formulations generally comprisebetween about 0.01% and about 95%, or between about 0.1% and about 90%,or between about 0.5% and about 90%, by weight of active substances.

In one embodiment, the active compound(s) have a purity of from about90% to 100%, or from about 95% to 100% (according to NMR spectrum).

As it pertains particularly to formulations of isolated HR elicitorprotein or polypeptide fragments, stable liquid compositions containinga harpin protein or polypeptide may involve obtaining a liquid extractthat is substantially free of cellular debris. In one embodiment, thisis accomplished by fermenting a suspension of harpin protein orpolypeptide-producing plant bacteria. Harpin protein or polypeptides canbe produced readily through fermentation in rapidly growing bacteria.For example, recombinant Escherichia coli may be used for large-scaleharpin protein or polypeptide production. Current technology enables theproduction of relatively large intracellular concentrations of harpinproteins or polypeptides.

Recombinant methodologies generally involve inserting a DNA moleculeexpressing a protein or polypeptide of interest into an expressionsystem to which the DNA molecule is heterologous (i.e., not normallypresent). The heterologous DNA molecule is inserted into the expressionsystem or vector in proper sense orientation and correct reading frame.The vector contains the necessary elements for the transcription andtranslation of the inserted protein-coding sequences. Transcription ofDNA is dependent upon the presence of a promoter. Similarly, translationof mRNA in prokaryotes depends upon the presence of the properprokaryotic signals which differ from those of eukaryotes. For a reviewon maximizing gene expression, see Roberts and Lauer, Methods inEnzymology 68:473 (1979), which is hereby incorporated by reference.

Regardless of the specific regulatory sequences employed, the DNAmolecule is cloned into the vector using standard cloning procedures inthe art, as described by Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, N.Y.(1989), which is hereby incorporated by reference in its entirety. Oncethe isolated DNA molecule encoding the harpin protein or polypeptide hasbeen cloned into an expression system, it is ready to be incorporatedinto a host cell. Such incorporation can be carried out by the variousforms of transformation, depending upon the vector/host cell system.Suitable host cells include, but are not limited to, bacteria, virus,yeast, mammalian cells, insect, plant, and the like.

Optionally, the recombinant host cells can be host cells that express anative or recombinant, functional type III secretion system. This isdescribed in detail in U.S. Pat. No. 6,596,509 to Bauer et al., which ishereby incorporated by reference in its entirety. As a consequence ofexpressing the functional type III secretion system, the cells willexpress the harpin protein or polypeptide and then secrete the proteininto the culture medium. This can simplify isolation and purification ofthe harpin protein or polypeptide.

The recombinant host cells can be grown in appropriate fermentationchambers, preferably under temperature and nutrient conditions thatoptimize growth of the host cells and the expression of the harpinproteins or polypeptides. Persons of skill in the art are fully able toidentify optimal conditions for particular host cells.

After fermentation, the bacterial suspension may be diluted in, e.g.,about 2 to 5 fold volume of a buffer to adjust the pH between about 5.5to 10, more preferably to a pH of between about 7 to 9, and even morepreferably to a pH of about 8.0. Suitable buffers are well-known in theart and may include, for example, potassium phosphate buffer or aTris-EDTA buffer. The concentration of the buffer can be from about0.001 mM to about 0.5 M.

Following the pH adjustment, the bacterial suspension solution is heattreated to a temperature of about 60-130° C., preferably to atemperature of about 95-125° C. Heat treatment may be carried out forany suitable period of time. In one embodiment, heat treatment iscarried out for a period of about five minutes up to about 30 minutes.

The heated suspension solution is then cooled. A suitable cool downtemperature is, without limitation, about 35-55° C., preferably about45° C.

Following cooling, bacterial cells in the bacterial suspension arelysed, if required, to liberate the harpin protein or polypeptide. Celllysis may be carried out, e.g., by contacting the bacterial suspensionwith a lysozyme. The concentration of lysozyme may be anywhere fromabout 2 ppm to 100 ppm. Alternatively, cell lysis may involvenon-chemical methods, such as high pressure or sonication, both of whichare well known by persons of ordinary skill in the art.

It may be desirable, after cell lysis, to incubate the bacterialsuspension. Suitable incubation times may vary. For example, it may bedesirable to incubate the bacterial suspension for a period of about30-45 minutes at a temperature of about 40-42° C.

After lysing, the desired protein or polypeptide (i.e., harpin proteinor polypeptide, or fragment thereof) can be further extracted byremoving the cell debris and the denatured proteins resulting from theprevious heat treatment step. In one embodiment, the extract iscentrifuged for about 10-20 minutes to remove some of the cell debris.Suitable centrifuge speeds may be from about 4,000 to 20,000 rpm and thespinning down time can be from about 10 minutes to 20 minutes. Furthercell debris may then be removed by heat treating and centrifuging thesupernatant to obtain a liquid extract that is substantially free ofcellular debris by removing more than about 60%, 70%, 80%, 90%, or 95%of total solids. This subsequent heat treatment may be carried out at atemperature of about 60° C. for up to about two hours, at about 100° C.for about 10 minutes, or at about 121° C. with 15 psi of pressure forabout 5 minutes. These temperatures and times may vary depending onother conditions.

A stable liquid composition containing a harpin protein or polypeptidemay further involve introducing into the liquid extract a biocidal agentand, optionally, one or both of a protease inhibitor and a non-ionicsurfactant, thereby obtaining a liquid composition comprising theisolated harpin protein or polypeptide fragment. In one embodiment, aprotease inhibitor is introduced into the liquid extract without anon-ionic surfactant. In another embodiment, a non-ionic surfactant isintroduced into the liquid extract without a protease inhibitor. In afurther embodiment, both a protease inhibitor and a non-ionic surfactantare introduced into the liquid extract. In yet another embodiment,neither a protease inhibitor nor a non-ionic surfactant are introducedinto the liquid extract.

Biocidal agents are added to the liquid extract for preservation.Suitable biocidal agents include, without limitation, antibiotics, toxicchemicals, and disinfectants. For example, a suitable antibiotic isstreptomycin, a suitable toxic agent is sodium azide, and a suitabledisinfectant is a Triple Action disinfectant (i.e., the EPA approvedpesticide with the following active ingredients: 1-decanaminium,N,N-dimethyl-N-octyl-, chloride (12.4% by mass); 1-octanaminium,N,N-dimethyl-N-octyl-, chloride (12.4% by mass);alkyl(C12-16)dimethylbenzylammonium chloride (12.4% by mass); sodiumcarbonate (3% by mass); and edentate sodium (2.5% by mass)). Theconcentration of biocidal agent introduced may be in the range of about1 ppm to about 100 ppm, more preferably about 2 ppm to about 30 ppm,most preferably about 5 ppm to about 10 ppm.

Protease inhibitors may be added to prevent harpin degradation byresidual proteases in the harpin extract. Protease inhibitors includevarious inhibitors classed by protease type or by their mechanism ofaction. Suitable protease inhibitors may include, without limitation,cysteine protease inhibitors, serine protease inhibitors (serpins),trypsin inhibitors, threonine protease inhibitors, aspartic acidprotease inhibitors, and metalloprotease inhibitors. Suitable proteaseinhibitors may be selected according to their mechanism of action. Forexample, suitable protease inhibitors may include, without limitation,suicide inhibitors, transition state inhibitors, protein proteaseinhibitors, and chelating agents. Examples of commercially availableprotease inhibitors include, without limitation, aprotinin, bestatin,calpain inhibitor I, calpain inhibitor II, chymostatin, E-64, leupeptin(N-acetyl-L-leucyl-L-leucyl-L-argininal), alpha-2-macroglobuline,pefabloc SC, pepstatin, PMSF (phenylmethanesulfonyl fluoride), andtosyl-L-lysine chloromethyl ketone (TLCK).

Protease inhibitors may be added to the extract at a concentration ofabout 1 ppm to about 100 ppm, more preferably about 2 ppm to about 30ppm, most preferably about 5 ppm to about 10 ppm.

Suitable non-ionic surfactants include, without limitation, sorbitanfatty acid ester, glycerin fatty acid ester, fatty acid polyglyceride,fatty acid alcohol polyglycol ether, acetylene glycol, acetylenealcohol, oxyalkylene block polymer, polyoxyethylene alkyl ether,polyoxyethylene alkylaryl ether, polyoxyethylene styrylaryl ether,polyoxyethylene glycol alkyl ether, polyoxyethylene fatty acid ester,polyoxyethylene sorbitan fatty acid ester, polyoxyethylene glycerinfatty acid ester, polyoxyethylene hydrogenated castor oil, andpolyoxypropylene fatty acid ester.

Non-ionic surfactants may be added to the extract at a volume amount ofabout 0.005 to about 20%, more preferably about 0.01 to about 15%, mostpreferably about 0.05% to about 10%.

As a result of introducing the biocidal agent and, optionally, theprotease inhibitor and surfactant as described above, the compositioncan maintain its harpin activity for at least 72 hours and preferablymuch longer. Preferably, the composition retains harpin activity formore than about 5 days, 1 week, 2 weeks, 3 weeks, or 4 weeks, morepreferably at least about 2 to 3 months, and most preferably longer thanabout 4 to 6 months. As used herein, retention of harpin activity can bedetermined by comparing the activity of the aged liquid composition to arecently prepared liquid composition or to a prior assessment made onthe same composition. The activity can be measured by the effects of thecomposition on plants as assessed by the disease resistance, growthenhancement, stress resistance, etc., of the plants following challenge.Preferably, the compositions of the present invention retain (for morethan 72 hours) at least about 70% activity, more preferably at leastabout 70% to about 80% activity, and most preferably at least about 80%to 90% activity.

Active substances of the composition of the present invention (i.e., atleast thiophanate methyl and an isolated hypersensitive responseelicitor protein or polypeptide fragment) can be applied alone and insequence, or as a single composition, e.g., in the form of directlysprayable solutions, powders, suspensions, dispersions, emulsions, oildispersions, pastes, dustable products, materials for spreading, orgranules, by means of spraying, atomizing, dusting, spreading, brushing,immersing, or pouring. The application form depends on the intendedpurpose to ensure in each case the finest possible distribution of theactive compound(s).

Aqueous application forms can be prepared from emulsion concentrates,pastes, or wettable powders (e.g., sprayable powders and oildispersions) by adding water. To prepare emulsions, pastes, or oildispersions, the substances (i.e., active compounds), as such ordissolved in an oil or solvent, can be homogenized in water by means ofa wetter, tackifier, dispersant, or emulsifier. Alternatively, it ispossible to prepare concentrates composed of an active substance,wetter, tackifier, dispersant, or emulsifier and, if appropriate,solvent or oil, and such concentrates are suitable for dilution withwater.

The weight ratio of the individual compounds to the total compositionwill depend on the properties of the individual compounds (e.g., thespecific isolated hypersensitive response elicitor protein orpolypeptide fragment, and/or the presence of other compounds). Theactive substance concentrations in the ready-to-use formulations can bevaried within relatively wide ranges. In general, they are from about0.0001% to about 10%, or from about 0.001% to about 1% by weight. Thecompositions may also be used successfully in the ultra-low-volumeprocess (ULV), it being possible to apply compositions comprising over95% by weight of active substances, or even to apply the activesubstances without additives.

Various types of oils, wetters, adjuvants, herbicides, fungicides, otherpesticides, or bactericides may be added to the agriculturalcompositions and, if appropriate, not until immediately prior to use(e.g., tank mix). These agents can be admixed with the active compoundsin a weight ratio of about 1:100 to about 100:1, or about 1:10 to about10:1. Individual pesticide components may be generally known by personsof ordinary skill in the art, such as those described in The PesticideManual, 15th Edition, British Crop Protection Council (2009), which ishereby incorporated by reference in its entirety.

The formulations may also contain fertilizers, such as ammonium nitrate,urea, potash, superphosphate, phytotoxicants, plant growth regulators,and safeners. Fertilizers may be applied separately from theformulations of the active substances or may be included in thecompositions, e.g., by being added immediately prior to use (tank mix).In one embodiment, a plant and/or area of cultivation may be sprayedwith a composition of the present invention either before or after thesame is treated with a fertilizer.

In one embodiment, the composition includes thiophanate methyl and isapplied to a plant and/or area of cultivation at an amount of betweenabout 1 fl oz/acre to about 200 fl oz/acre of thiophanate methyl, orbetween about 5 fl oz/acre and about 100 fl oz/acre of thiophanatemethyl, or between about 10 fl oz/acre and about 50 fl oz/acre ofthiophanate methyl, or between about 15 fl oz/acre to about 30 floz/acre of thiophanate methyl.

In another embodiment, the composition includes an isolatedhypersensitive response elicitor protein or polypeptide fragment and isapplied to a plant and/or area of cultivation at an amount of betweenabout 0.25 dry oz/acre and about 15 dry oz/acre of the isolatedhypersensitive response elicitor protein or polypeptide fragment, orbetween about 0.5 dry oz/acre and about 10 dry oz/acre of the isolatedhypersensitive response elicitor protein or polypeptide fragment, orbetween about 0.75 dry oz/acre and about 5 dry oz/acre of the isolatedhypersensitive response elicitor protein or polypeptide fragment, orbetween about 1 dry oz/acre to about 2.5 dry oz/acre of the isolatedhypersensitive response elicitor protein or polypeptide fragment.

Active compounds in the formulations may have different crystalmodifications to alter biological activity. In all ternary andquaternary compositions used, compounds are present in amounts whichresult in a synergistic plant health increasing effect.

Further active compounds, e.g., insecticides, herbicides, fungicides,and/or herbicidal or growth-regulating compounds or fertilizers can thenbe added as further active components according to need. In oneembodiment, formulations of the present invention may include one ormore additional active ingredients selected from the group ofglyphosate, dicamba, and glufosinate. In this embodiment, the plants areresistant to these herbicides.

Glyphosate and dicamba can also be used as their agriculturallyacceptable salts and esters. Suitable salts of glyphosate include thosesalts of glyphosate where the counterion is an agriculturally acceptablecation. Suitable non-limiting examples of such salts areglyphosate-ammonium, glyphosate-diammonium, glyphosate-dimethylammonium,glyphosate-isopropylammonium, glyphosate-potassium, glyphosate-sodium,glyphosate-sesquisodium, glyphosate-sesquipotassium,glyphosate-trimethylsulphonium (sulphosate), glyphosate-trimesium, aswell as the ethanolamine and diethanolamine salts. In one embodiment,the salt of glyphosate is selected from glyphosate-diammonium,glyphosate-isopropylammonium, glyphosate-sesquisodium, andglyphosata-trimethylsulphonium (sulphosate).

Suitable salts of dicamba include those salts of dicamba where thecounterion is an agriculturally acceptable cation. Suitable examples ofsuch salts are dicamba-sodium, dicamba-potassium,dicamba-methylammonium, dicamba-dimethylammonium,dicamba-isopropylammonium, dicamba-diglycolamine, dicamba-olamine,dicamba-diolamine, and dicamba-trolamine Examples of a suitable esterare dicamba-methyl and dicamba-butoyl.

For a composition that includes glyphosate, application of thecomposition to a plant, plant part, area of cultivation, or anycombination thereof is, according to one embodiment, carried out at arate of between about 1 g/ha and about 2500 g/ha of glyphosate, orbetween about 5 g/ha and about 1500 g/ha glyphosate, or between about100 g/ha and about 750 g/ha of glyphosate.

For a composition of the present invention that includes dicamba,application of the composition to a plant, plant part, area ofcultivation, or any combination thereof is, according to one embodiment,carried out at a rate of between about 1 g/ha and about 1500 g/ha, orbetween about 5 g/ha and about 750 g/ha, or between about 50 g/ha andabout 500 g/ha.

For a composition of the present invention that includes glufosinate,application of the composition to a plant, plant part, area ofcultivation, or any combination thereof is, according to one embodiment,carried out at a rate of between about 1 g/ha and about 1000 g/ha, orbetween about 5 g/ha and about 500 g/ha, or between about 20 g/ha andabout 300 g/ha, or between about 30 g/ha and about 200 g/ha.

Applying thiophanate methyl and an isolated HR elicitor protein orpolypeptide fragment according to the method of the present inventioncan be carried out in the absence of pest (e.g., fungal pathogen)pressure and/or both before and after an infection of the materials orplants by any pest (e.g., fungal pathogen).

Applying thiophanate methyl and an isolated HR elicitor protein orpolypeptide fragment according to the method of the present inventionmay occur at various different growth stages of the plant depending onthe desired effect.

The term “growth stage” (GS) refers to the extended BBCH-scale, which isa system for a uniform coding of phenologically similar growth stages ofall mono- and dicotyledonous plant species in which the entiredevelopmental cycle of the plants is subdivided into clearlyrecognizable and distinguishable longer-lasting developmental phases.The BBCH-scale uses a decimal code system, which is divided intoprincipal and secondary growth stages. The abbreviation BBCH derivesfrom the Federal Biological Research Centre for Agriculture and Forestry(Germany), the Bundessortenamt (Germany) and the chemical industry.

Applying may be carried out at a growth stage (GS) between GS 00 and GS73 BBCH, GS00 and GS 63 BBCH, GS11 and GS 63 BBCH, or GS 11 and GS 34BBCH.

Applying may be carried out repeatedly, e.g., in 2, 3, 4, 5, 6, 7, 8, ormore applications. In one embodiment, thiophanate methyl and an isolatedHR elicitor protein or polypeptide fragment are applied first betweenBBCH growth stages 11 to 63 and again between BBCH growth stages 65 to75.

Applying thiophanate methyl and an isolated HR elicitor protein orpolypeptide fragment may be carried out by foliar spray treatment,in-furrow application, or by any other means.

According to the present invention, simultaneous (i.e., joint orseparate) application of thiophanate methyl and an isolatedhypersensitive response elicitor protein or polypeptide fragment (and,optionally, an additional active compound) and successive application ofthiophanate methyl and an isolated hypersensitive response elicitorprotein or polypeptide fragment (and, optionally, an additional activecompound) enhance and/or increase the health of a plant compared to whatis possible by applying either thiophanate methyl or an isolatedhypersensitive response elicitor protein or polypeptide fragment alone.The active compounds thiophanate methyl and an isolated hypersensitiveresponse elicitor protein or polypeptide fragment have a synergisticeffect on plant yield, meaning that they are used in a quantity whichgives the desired effect, which is a synergistic increase of the healthof a plant but which preferably does not give rise to any phytotoxicsymptom on the treated plant.

In carrying out the method of the present invention, the overall healthof a plant is increased due the synergistic effect of applyingthiophanate methyl and an isolated hypersensitive response elicitorprotein or polypeptide fragment to a plant and/or area of cultivation. Asynergistic effect is where the purely additive effect (in mathematicalterms) of the application of the individual components is surpassed bythe application of the combination (see FIGS. 1-2).

As used herein, “health of a plant” or “plant health” means thecondition of a plant and/or its products which is determined by severalaspects alone or in combination with each other, such as increasedyield, plant vigor, quality, and tolerance to abiotic and/or bioticstress.

A plant suffering from fungal or insecticidal attack often produces asmaller biomass, which leads to a reduced yield as compared to a plantwhich has been subjected to curative or preventive treatment against thepathogenic fungus or any other relevant pest and which can grow withoutthe damage caused by the biotic stress factor. However, applyingthiophanate methyl and an isolated hypersensitive response elicitorprotein or polypeptide fragment pursuant to the method of the presentinvention leads to enhanced plant health even in the absence of anybiotic stress. This means that the positive effects of thiophanatemethyl and an isolated hypersensitive response elicitor protein orpolypeptide fragment cannot be explained just by the fungicidal,insecticidal, and/or herbicidal activities of the active ingredients,but is based on further activity profiles. As a result, application ofthiophanate methyl and an isolated hypersensitive response elicitorprotein or polypeptide fragment to a plant and/or area of cultivationcan also be carried out in the absence of pest pressure on the plant.

According to the present invention, “increasing yield of a plant” meansthat the yield of a product of the plant is increased by a measurableamount over the yield of the same product of the plant produced underthe same conditions, but without application of both thiophanate methyland an isolated hypersensitive response elicitor protein or polypeptidefragment to the plant and/or area of cultivation. In one embodiment, theterm “yield” refers to fruits in the proper sense, as well asvegetables, nuts, grains, and seeds.

“Grain” and “fruit” are to be understood as any plant product which isfurther utilized after harvesting, e.g., fruits in the proper sense,vegetables, nuts, grains, seeds, wood (e.g., in the case of silvicultureplants), flowers (e.g., in the case of gardening plants andornamentals), etc., meaning anything of economic value that is producedby the plant.

Increased yield of a plant can be characterized by the followingnon-limiting properties: increased plant weight; increased biomass, suchas higher overall fresh weight (FW) and/or higher overall dry weight(DW); increased number of flowers per plant; higher grain and/or fruityield; more tillers or side shoots (branches); larger leaves; increasedshoot growth; increased protein content; increased oil content;increased starch content; increased pigment content; increasedchlorophyll content; and any combination thereof.

Chlorophyll content has a positive correlation with a plant'sphotosynthesis rate and, accordingly, the higher the chlorophyll contentthe higher the yield of a plant.

According to one embodiment, a synergistic increase in yield in drybeans is greater than about 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, ormore compared to plants where no thiophanate methyl and isolatedhypersensitive response elicitor protein or polypeptide fragment isapplied to the plant and/or area of cultivation.

According to another embodiment, a synergistic increase in yield inpeanut is greater than about 2%, 3%, 4%, or more compared to plantswhere no thiophanate methyl and isolated hypersensitive responseelicitor protein or polypeptide fragment is applied to the plant and/orarea of cultivation.

Increasing the yield of a plant may involve improving plant vigor. Plantvigor becomes manifest in several aspects, including the general visualappearance of the plant. Improved plant vigor can be characterized by,inter alia, the following: improved vitality of the plant; improvedplant growth; improved plant development; improved visual appearance;improved plant stand (less plant verse/lodging); improved emergence;enhanced root growth and/or more developed root system; enhancednodulation, in particular rhizobial nodulation; bigger leaf blade;bigger size; increased plant height; increased tiller number; increasednumber of side shoots; increased number of flowers per plant; increasedshoot growth; increased root growth (extensive root system); enhancedphotosynthetic activity; enhanced pigment content; earlier flowering;earlier fruiting; earlier and improved germination; earlier grainmaturity; fewer non-productive tillers; fewer dead basal leaves; lessinput needed (such as fertilizers or water); greener leaves; completematuration under shortened vegetation periods; less fertilizer needed;fewer sowing of seeds needed; easier harvesting; faster and more uniformripening; longer shelf-life; longer panicles; delay of senescence;stronger and/or more productive tillers; better extractability ofingredients; improved quality of seeds (for being seeded in thefollowing seasons for seed production); reduced production of ethyleneand/or the inhibition of its reception by the plant; and any combinationthereof.

Enhanced photosynthetic activity of a plant may be based on increasedstomatal conductance and/or an increased CO₂ assimilation rate of theplant.

Increasing the yield of a plant may involve improving the quality of aplant and/or its products. Improvements in plant quality may include,without limitation, improving certain plant characteristics, such asincreasing the content and/or composition of certain ingredients by ameasurable or noticeable amount over the same factor of the plantproduced under the same conditions, but without application of thecomposition of the present invention. Enhanced quality can becharacterized by, inter alia, the following: increased nutrient content;increased protein content; increased content of fatty acids; increasedmetabolite content; increased carotenoid content; increased sugarcontent; increased amount of essential amino acids; improved nutrientcomposition; improved protein composition; improved composition of fattyacids; improved metabolite composition; improved carotenoid composition;improved sugar composition; improved amino acids composition; improvedor optimal fruit color; improved leaf color; higher storage capacity;higher processability of the harvested products; or any combinationthereof.

Increasing the yield of a plant may involve improving a plant'stolerance or resistance to biotic and/or abiotic stress factors. Bioticand abiotic stress, especially over longer terms, can have harmfuleffects on plants. Biotic stress is caused by living organisms whileabiotic stress is caused, for example, by environmental extremes. In oneembodiment, applying the composition of the present invention to a plantpursuant to the method of the present invention enhances tolerance orresistance to biotic and/or abiotic stress factors, meaning: (1) certainnegative factors caused by biotic and/or abiotic stress are diminishedin a measurable or noticeable amount as compared to plants exposed tothe same conditions, but without being treated with an inventive mixtureand (2) the negative factors are not diminished by a direct action ofthe composition on the stress factors, e.g., by its fungicidal orinsecticidal action which directly destroys the microorganisms or pests,but rather by a stimulation of the plants' own defensive reactionsagainst said stress factors.

Negative factors caused by biotic stress, such as pathogens and pests,are widely known and range from dotted leaves to total destruction ofthe plant. Biotic stress can be caused by living organisms, such aspests (e.g., insects, arachnides, and nematodes), competing plants(e.g., weeds), microorganisms (e.g., phytopathogenic fungi and/orbacteria), and/or viruses.

Negative factors caused by abiotic stress are also well-known and canoften be observed either as reduced plant vigor (as described above) orby the following symptoms: dotted leaves, “burned” leaves, reducedgrowth, fewer flowers, less biomass, less crop yield, reducednutritional value of the crop, and later crop maturity, to give just afew examples. Abiotic stress can be caused by, inter alia: extremes intemperature such as heat or cold (heat stress/cold stress), strongvariations in temperature, temperatures unusual for the specific season,drought (drought stress), extreme wetness, high salinity (salt stress),radiation (e.g., by increased UV radiation due to the decreasing ozonelayer), increased ozone levels (ozone stress), organic pollution (e.g.,by phytotoxic amounts of pesticides), inorganic pollution (e.g., byheavy metal contaminants), and any combination thereof.

Biotic and/or abiotic stress factors decrease the quantity and thequality of the stressed plants, their crops, and fruits. As far asquality is concerned, reproductive development can be affected withconsequences on the crops which are important for fruits or seeds.Synthesis, accumulation, and storage of proteins are mostly affected bytemperature; growth is slowed by almost all types of stress;polysaccharide synthesis, both structural and storage, is reduced ormodified. These effects result in a decrease in biomass (yield) and inchanges in the nutritional value of the plant product.

The above identified indicators for the health condition of a plant maybe interdependent and may result from each other. For example, anincreased resistance to biotic and/or abiotic stress may lead to abetter plant vigor, e.g., to better and bigger crops, and thus to anincreased yield. Inversely, a more developed root system may result inan increased resistance to biotic and/or abiotic stress.

Applying thiophanate methyl and an isolated hypersensitive responseelicitor protein or polypeptide fragment to a plant and/or area ofcultivation has a synergistic effect on the plant to: increase thehealth of the plant, increase the yield of the plant, increase thebiomass of the plant, increase the oil content of the plant, increasethe vigor of the plant, increase the stand of the plant, increase theemergence of the plant, increase the root growth of the plant, increasethe photosynthetic activity of the plant, improve the quality of theplant, improve the nutrient composition of the plant, improve theprotein composition of the plant, improve the carotenoid composition ofthe plant, increase the tolerance of the plant to biotic stress,increase the tolerance of the plant to fungi, increase the tolerance ofthe plant to nematodes, increase the tolerance of the plant to bacteria,increase the tolerance of the plant to abiotic stress, increase thetolerance of the plant to drought stress, increase the tolerance of theplant to cold stress, increase the tolerance of the plant to heatstress, increase the tolerance of the plant to salt stress, increase thetolerance of the plant to ozone stress, and/or any combination thereof.

One of the most important factors for increased resistance againstbiotic and abiotic stress is the stimulation of the plant's naturaldefense reactions, which occurs by application of thiophanate methyl andan isolated hypersensitive response elicitor protein or polypeptidefragment according to the method of the present invention.

It is also possible for thiophanate methyl and an isolatedhypersensitive response elicitor protein or polypeptide fragment (and,optionally, additional active compounds) to be packaged and used as acombination composition. Thus, another aspect of the present inventionrelates to a kit comprising formulated thiophanate methyl and anisolated hypersensitive response elicitor protein or polypeptidefragment (and, optionally, additional active compounds).

In one embodiment, the kit may include one or more, including all,components that may be used to prepare an agrochemical compositioncontaining thiophanate methyl and an isolated hypersensitive responseelicitor protein or polypeptide fragment (and, optionally, additionalactive compounds). For example, the kit may include thiophanate methyland an isolated hypersensitive response elicitor protein or polypeptidefragment (and, optionally, an additional active compound) and/or anadjuvant component and/or a further pesticidal compound (e.g.,insecticide, fungicide, or herbicide) and/or a growth regulatorcomponent. One or more of the components may already be combinedtogether or pre-formulated.

In those embodiments where more than two active compounds are providedin a kit, the active compounds may already be combined together and assuch are packaged in a single container such as a vial, bottle, can,pouch, bag, or canister. In an alternative embodiment, two or moreactive compounds may be packaged separately (i.e., not pre-formulated).As such, kits may include one or more separate containers such as vials,cans, bottles, pouches, bags, or canisters, each container containing aseparate component for an agrochemical composition. In both forms, acomponent of the kit may be applied separately from or together with thefurther components or as a component of a combination composition.

The user may then apply the composition(s) usually from a predosagedevice, a knapsack sprayer, a spray tank, or a spray plane. Here, theagrochemical composition is made up with water and/or buffer to thedesired application concentration, it being possible, if appropriate, toadd further auxiliaries, and the ready-to-use spray liquid or theagrochemical composition according to the invention is thus obtained. Inone embodiment, about 50 to about 500 liters of the ready-to-use sprayliquid are applied per hectare of agricultural useful area, or about 50to about 400 liters.

In another embodiment, either individual compounds formulated as acomposition or partially premixed compounds (e.g., thiophanate methyland an isolated hypersensitive response elicitor protein or polypeptidefragment (and, optionally, an additional active compound)) are mixed bythe user in a spray tank and further auxiliaries and additives areadded, if appropriate (tank mix).

In another embodiment, individual compounds (e.g., thiophanate methyland an isolated hypersensitive response elicitor protein or polypeptidefragment (and, optionally, an additional active compound)) or partiallypremixed compounds (e.g. thiophanate methyl and an isolatedhypersensitive response elicitor protein or polypeptide fragment (and,optionally, an additional active compound)) are applied jointly (e.g.,after tank mix) or consecutively.

These aspects of the present invention are further illustrated by theexamples below.

EXAMPLES

The following examples are provided to illustrate embodiments of thepresent invention, but they are by no means intended to limit its scope.

Example 1 Control of White Mold in Pinto Bean Plants with PHC-351 andThiophanate Methyl

The objective of this Example is to provide answers to the followingquestions: When applied at the specific timing, does PHC-351 affect thevisual appearance of the crop? Does PHC-351 affect yield when applied atthe specified timing? Does PHC-351 reduce white mold (Sclerotiniasclerotiorum) incidence or severity when applied at the specifiedtiming? Is there an interaction between Topsin® M 70WP and PHC-351 interms of white mold (Sclerotinia sclerotiorum) management and overallplant yield?

Materials

PHC-351 contains the harpin fusion protein of SEQ ID NO:1, which is aplant elicitor. In this trial, it was tested as a foliar spray on drybean plants, in combination with Topsin® M treatment. Topsin® M 70WP isa thiophanate-methyl fungicide 70% Wettable Powder. Previous trialsindicate that the timing specified in this protocol is later thandesirable for influencing crop set. To influence plant growth and yield,harpin is normally applied at an earlier stage of plant development, thevegetative phase, which is prior to the development of fruiting bodies.Here it was applied at a later stage where the fruiting body was alreadyin existence.

Plot Establishment and Test Layout

The test was initiated on a number of test sites suitable for dry beanproduction, and likely to have white mold. The sites had dry beans withwhite mold the previous year, and was not plowed the year of the test.Pinto varieties with a tolerance for white mold (such as ‘Chase’) wereavoided.

Plot size, row spacing, and buffers: 6 rows (15-30″ spacing=10 ftwide)×25+ ft plots. At least 5 ft down row between blocks. The 2 outerrows on both sides of the plot were buffers or borders that were notharvested.

Trial design: Plant 8 reps. Use a Randomized complete block design.After the 14 day stand count, block the plots based on stand.

Planting Date: Seed was planted within a commercially acceptableplanting window.

Plant Density: This trial emphasizes yield; consequently, planting wasto achieve highly uniform plant spacing down the row.

Site Management: Plant growth (fertilizer), insects, diseases, and weedswere managed according to locally accepted practices and within theother limitations mentioned in this protocol. All plots were managedidentically, and test sites were managed to grow healthy plants whileminimizing harmful impact of pests.

Mixing and Application

Spray Application: Treatments were applied with properly calibratedground equipment equipped with a shielded spray boom to minimize spraydrift. With 5 ft wide, 2-row spray boom having 3 nozzles spaced ˜18-20inches apart, the center two rows of each plot were sprayed. Boom widthwas adjusted to fit row spacing.

Rinsing: All mixing and application equipment was carefully triplerinsed prior to spraying plots and spray system was triple rinsed beforeswitching to the next treatment. Spray treatments were carried out inthe order of the Treatment List (Table 4).

Water Volume and Source: 20-30 gpa. Sprayed to ensure thorough coverageof the blooms, using drop nozzles if available. If tank water was highin total mineral content, salinity, and/or suspended solids, clean waterwas used.

Tank Mixtures: PHC-351 was applied to appropriate plots and treatmentswere allowed to dry. Then, Topsin® M 70WP was applied to appropriateplots. No other materials were added to these treatments.

PHC-351 Handling: Product was used within 8 hours of mixing. Forreplicated trial purposes: opened packets were used within 8 hrs. Afteropening, what was needed for the application was used, and the remainingmaterial was discarded. Sprays were made on a day and at a time when theplants were actively growing.

Treatment Timing: Application was made to achieve thorough coverage ofthe blooms at the timing specified in treatment list, i.e., when 100% ofthe plants had at least 1 open bloom.

TABLE 4 Treatment List Treatment Rate Rate Unit Timing 1 Water Applywhen 100% of the plants have 1 Control* open bloom 2 PHC-351 1 Oz/acreApply when 100% of the plants have 1 open bloom 3 Topsin ® M 1.5 lb/acreApply when 100% of the plants have 1 open bloom 4 PHC-351 1 Oz/acreApply when 100% of the plants have 1 4 Topsin ® M 1.5 lb/acre open bloom*The water control should be treated with water at the same time as thechemical treatments.

Data Collection

At Planting—Composite Soil Sample: A composite soil sample was collectedand analyzed 0-7 days before planting. The sample was intended tocharacterize the entire trial site. 20 soil cores were extractedrandomly from across the planting area of the field trial and cores weremixed. The soil cores were submitted to an accredited lab and requestedanalysis for Organic Matter, micronutrients, macronutrients, soil pH,calculated Cation Exchange Capacity, and % Cation Saturation. Thesamples were handled as per standard soil analysis guidelines.

In-Season Counts: These measurements were requested to determine ifthere were early visual effects associated with the treatments.

(a) Plant stand counted at 14 and 28 Days After Emergence (DAE), countedentire length of two rows, then converted and reported as plants/acre.Used the 14 day count to block replicates, that is, had the same ornearly the same stand count for all entries within a replicate.

(b) Vigor (1-9) at 14 and 28 DAE by visually estimating vigor.

(c) Percent canopy closure at bloom, recorded for center two rows.

Disease Incidence and Severity: 10-14 days before harvest both theincidence of white mold in percent infected plants, and the severitywere rated on a 1-5 scale, where 1 is the least severe and 5 is the mostsevere.

Yield: The center 2 rows of each plot were machine harvested. Harvestwas within customary window and harvest delays were avoided. To minimizeedge effects, harvesting was avoided the first and last 2.5 feet of eachplot. Report was made of bean weight per plot and moisture content (%).After correcting for moisture, yield was calculated and reported asbushels per acre (bu/A).

Statistical Analysis: ANOVA and Fishers Protected LSD, P=0.10.

Experimental results are reported in FIG. 1.

The trials were completed in dry bean production regions where whitemold is frequently a problem. 2012 was hotter and drier than normal at 6of the sites, and colder and wetter than normal at the remaining sites.Pre-spray data showed no difference in stand, vigor, or canopy closure,indicating no unusual pre-existing plot variability. White mold appearedat 5 of the sites (in WA, WI, MI, & ID). Overall when PHC-351 (Harpin)was applied alone it increased the yield by an average of 3.59% over theuntreated control. When Topsin® M (Thiophanate Methyl) was applied aloneit increased the yield by 18.6% over the untreated control. However,when they were both used on the plant, the yield increased by more thanthe sum of 3.59% and 18.6%. The yield increase for the combination was25.1%; this is nearly 3% more than what would be predicted,demonstrating an unexpected and synergistic result. Similar resultswould be expected for any other dry bean variety.

Example 2 Influence on Yield and Leaf Spot Control in Runner-Type PeanutPlants with PHC-351 and Thiophanate Methyl

The objective of this Example is to provide answers to the followingquestions: When applied at the specific timing, does PHC-351 affect thevisual appearance of the crop? Does PHC-351 affect yield when applied atthe specified timing? Does PHC-351 reduce white mold (Early=Cercospora,Late=Cerosporidium) incidence or severity when applied at the specifiedtiming? Is there an interaction between Topsin® M and PHC-351 in termsof white mold (Early=Cercospora, Late=Cerosporidium) management andoverall plant yield?

Materials

PHC-351 contains the harpin fusion protein of SEQ ID NO:1, which is aplant elicitor. In this trial, it was tested as a foliar spray onRunner-Type peanut plants, in combination with Topsin® M treatment.Topsin® M 70WP is a thiophanate-methyl fungicide 70% Wettable Powder.Previous trials indicate that the timing specified in this protocol islater than desirable for influencing crop set. To influence plant growthand yield, harpin is normally applied at an earlier stage of plantdevelopment, the vegetative phase, which is prior to the development offruiting bodies. Here it was applied at a later stage where the fruitingbody was already in existence.

Plot Establishment and Test Layout

The test was initiated on a test site suitable for Runner Type peanutproduction, and likely to have leaf spot.

Plot size, row spacing, and buffers: 6 rows (36″ spacing=18 ft wide)×30+ft plots. At least 10 ft alley was left between blocks. The 2 outer rowson both sides of the plot were borders that were not treated orharvested.

Trial design: Planted 6 reps. Used a Randomized complete block design.The plots were blocked based on uniform stand.

Planting Date: Seed was planted within a commercially acceptableplanting window.

Plant Density: This trial emphasizes yield; consequently, planting wasto achieve highly uniform plant spacing down the row.

Site Management: Insects, diseases, and weeds were managed according tolocally accepted practices and within the other limitations mentioned inthis protocol. All plots were managed identically, and test site wasmanaged to grow healthy plants while minimizing harmful impact of pests.The entire trial area was sprayed with other fungicides (chlorothalonil)to control diseases.

Mixing and Application

Spray Application: Treatments were applied with properly calibratedground equipment equipped with a shielded spray boom to minimize spraydrift. With 6 ft wide, 2-row spray boom having 6 nozzles spaced ˜18-24inches apart, the center four rows of each plot were sprayed. Boom widthwas adjusted to fit row spacing.

Rinsing: All mixing and application equipment was carefully triplerinsed prior to spraying plots and spray system was triple rinsed beforeswitching to the next treatment. Spray treatments were carried out inthe order of the Treatment List (Table 5).

TABLE 5 Treatment List Rate Treatment Rate Unit Timing 1 Water Apply at35-40 DAE and repeat on 14 day Control* intervals as needed for leafspot control. Apply at least 3 applications. 2 PHC-351 1 Oz/acre Applyat 35-40 DAE and repeat on 14 day intervals as needed for leaf spotcontrol. Apply at least 3 applications. 3 Topsin ® M 0.5 lb/acre Applyat 35-40 DAE and repeat on 14 day intervals as needed for leaf spotcontrol. Apply at least 3 applications. 4 PHC-351 1 Oz/acre Apply at35-40 DAE and repeat on 14 day intervals as needed for leaf spotcontrol. Apply at least 3 applications. 4 Topsin ® M 0.5 lb/acre Applyat 35-40 DAE and repeat on 14 day intervals as needed for leaf spotcontrol. Apply at least 3 applications. *The water control should betreated with water at the same time as the chemical treatments.

Water Volume and Source: 15-20 gpa. Sprayed to ensure thorough coverageof the foliage. If tank water was high in total mineral content,salinity, and/or suspended solids, clean water was used.

Tank Mixtures: PHC-351 was applied to appropriate plots and treatmentswere allowed to dry. Then, Topsin® M was applied to appropriate plots.No other materials were added to these treatments.

PHC-351 Handling: Product was used within 8 hours of mixing. Forreplicated trial purposes: opened packets were used within 8 hrs. Afteropening, what was needed for the application was used, and the remainingmaterial was discarded. Sprays were made on a day and at a time when theplants were actively growing.

Treatment Timing: Application was made 35-40 DAE and repeated on 14 dayintervals as needed for leaf spot control. At least 3 applications weremade. No more than 2 lbs./A of Topsin® were applied per season.

Data Collection

At Planting—Composite Soil Sample: A composite soil sample was collectedand analyzed 0-7 days before planting. The sample was intended tocharacterize the entire trial site. 20 soil cores were extractedrandomly from across the planting area of the field trial and cores weremixed. The soil cores were submitted to an accredited lab and requestedanalysis for Organic Matter, micronutrients, macronutrients, soil pH,calculated Cation Exchange Capacity, and % Cation Saturation. Thesamples were handled as per standard soil analysis guidelines.

In-Season Counts: These measurements were requested to determine ifthere were early visual effects associated with the treatments.

(a) Plant stand counted at 28 Days After Emergence (DAE), counted entirelength of two rows, then converted and reported as plants/acre. Used the28 day count to block replicates, that is, had the same or nearly thesame stand count for all entries within a replicate.

(b) Vigor (1-9) at 28 DAE by visually estimating vigor for entire plot(1=poorest and 9=best).

Disease Incidence and Severity: Leaf spot was rated at 30 days beforeharvest and again at 5-7 days before harvest. Both the incidence of LeafSpot (Early=Cercospora, Late=Cerosporidium) in percent infected plants,and the severity on a 1-10 scale was reported, where 1 is the leastsevere and 10 is the most severe.

Yield: The center 2 rows of each plot were machine harvested. Harvestwas within customary window and harvest delays were avoided. Report wasmade of peanut weight per plot and yield was calculated and reported aspounds per acre (lbs/A).

Statistical Analysis: ANOVA and Fishers Protected LSD, P=0.10.

Experimental results are reported in FIG. 2.

The trials were conducted according to the protocol as described above.The disease pressure (Late Leaf Spot Mycosphaerella berkeleyi) was lowto moderate in these trials. Yields were recorded and were noted to bevery high and similar in the two locations. The PHC-351 treatment wasshown to reduce yield slightly (by 5.3% or 311 lbs). However, theTopsin® M (Thiophanate Methyl) treatment increased yield slightly; whenapplied alone Topsin® M increased yield by 3.0% over the untreatedcontrol (177 lbs). It was surprising to see that despite the fact thatPHC-351 caused a mild deleterious effect to yield when applied alone,when it was applied in combination with Thiophanate Methyl the yieldincreased by 3.7% over the untreated control (218 lb). The combinationincreased yield more than either of the products did separately, oradditively, displaying clear synergy.

Although the invention has been described in detail for the purpose ofillustration, it is understood that such detail is solely for thatpurpose, and variations can be made therein by those skilled in the artwithout departing from the spirit and scope of the invention which isdefined by the following claims.

What is claimed:
 1. A method of increasing yield of a plant, said methodcomprising: applying to a plant and/or area of cultivation thiophanatemethyl and an isolated hypersensitive response elicitor protein orpolypeptide fragment, wherein said applying is carried out underconditions effective to induce a synergistic yield from the plant. 2.The method according to claim 1, wherein yield is selected from improvedplant vigor, increased tolerance or resistance to biotic and/or abioticstress, increased plant weight, increased biomass, increased number offlowers per plant, higher grain and/or fruit yield, more tillers or sideshoots, larger leaves, increased shoot growth, increased proteincontent, increased oil content, increased starch content, increasedpigment content, increased chlorophyll content, and combinationsthereof.
 3. The method according to claim 1, wherein the plant is drybean and yield is increased by at least about 23%.
 4. The methodaccording to claim 1, wherein the plant is peanut and yield is increasedby at least about 3%.
 5. The method according to claim 1, whereinthiophanate methyl and an isolated hypersensitive response elicitorprotein or polypeptide fragment are applied as a single composition. 6.The method according to claim 1, wherein thiophanate methyl and anisolated hypersensitive response elicitor protein or polypeptidefragment are applied as separate compositions at the same time.
 7. Themethod according to claim 1, wherein thiophanate methyl and an isolatedhypersensitive response elicitor protein or polypeptide fragment areapplied as separate compositions successively.
 8. The method accordingto claim 1, wherein said applying further comprises: applying aneffective amount of an additional insecticide, an additional fungicide,an herbicide, a plant growth regulator, or combinations thereof.
 9. Themethod according to claim 8, wherein said applying comprises applying aneffective amount of an herbicide selected from glyphosate, dicamba,glufosinate, agriculturally acceptable salts and esters thereof, or acombination thereof.
 10. The method according to claim 1, wherein saidapplying is carried out by spraying, atomizing, dusting, spreading,brushing, immersing, and/or pouring.
 11. The method according to claim1, wherein said applying is at an amount of between about 1 fl oz/acreto about 200 fl oz/acre of thiophanate methyl.
 12. The method accordingto claim 1, wherein said applying is at an amount of between about 0.25dry oz/acre to about 15 dry oz/acre of the hypersensitive responseelicitor protein or polypeptide fragment.
 13. The method according toclaim 1, wherein the hypersensitive response elicitor protein orpolypeptide fragment is recombinant.
 14. The method according to claim1, wherein the hypersensitive response elicitor protein or polypeptidefragment comprises a full-length hypersensitive response elicitorprotein.
 15. The method according to claim 1, wherein the hypersensitiveresponse elicitor protein or polypeptide fragment comprises a fusionprotein.
 16. The method according to claim 15, wherein said fusionprotein comprises an isolated pair or more of spaced apart domains, eachcomprising an acidic portion linked to an alpha-helix.
 17. The methodaccording to claim 16, wherein each domain is from a different sourceorganism.
 18. The method according to claim 16, wherein there are 3 ormore spaced apart domains.
 19. The method according to claim 1, whereinthe plant is selected from legume, bean, peanut, celery, cucurbit,canola, wheat, stone fruit, apple, blueberry, strawberry, alfalfa, rice,barley, rye, cotton, sunflower, corn, potato, sweet potato, pea,chicory, lettuce, endive, cabbage, brussel sprout, beet parsnip,cauliflower, broccoli, turnip, radish, spinach, onion, garlic, eggplant,pepper, carrot, squash, pumpkin, zucchini, cucumber, pear, melon,citrus, grape, raspberry, pineapple, soybean, tobacco, tomato, sorghum,and sugarcane.
 20. The method according to claim 1, wherein the plant isselected from dry bean, peanut, pea, and soybean.
 21. A compositioncomprising a liquid or solid carrier, thiophanate methyl, and anisolated hypersensitive response elicitor protein or polypeptidefragment.
 22. The composition according to claim 21, wherein the carrieris a liquid carrier.
 23. The composition according to claim 21, whereinthe carrier is a solid carrier.
 24. The composition according to claim21, wherein the composition is in the form of a solution, emulsion,suspension, dust, powder, paste, or granule.
 25. The compositionaccording to claim 21 further comprising: an effective amount of anadditional insecticide, an additional fungicide, an herbicide, a plantgrowth regulator, or a combination thereof.
 26. The compositionaccording to claim 25, wherein the composition comprises an effectiveamount of an herbicide selected from glyphosate, dicamba, glufosinate,agriculturally acceptable salts and esters thereof, or a combinationthereof.
 27. The composition according to claim 21, wherein thehypersensitive response elicitor protein or polypeptide fragment isrecombinant.
 28. The composition according to claim 21, wherein thehypersensitive response elicitor protein or polypeptide fragmentcomprises a full-length hypersensitive response elicitor protein. 29.The composition according to claim 21, wherein the hypersensitiveresponse elicitor protein or polypeptide fragment comprises a fusionprotein.
 30. The composition according to claim 21, wherein thehypersensitive response elicitor protein or polypeptide fragmentcomprises an isolated pair or more of spaced apart domains, eachcomprising an acidic portion linked to an alpha-helix.
 31. Thecomposition according to claim 30, wherein each domain is from adifferent source organism.
 32. The composition according to claim 30,wherein there are 3 or more spaced apart domains.